AU771086B2 - Extracellular matrix signalling molecules - Google Patents

Description

-1-

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name of Applicant: Munin Corporation Actual Inventor: Lester F. Lau Address for Service: BALDWIN SHELSTON WATERS MARGARET STREET SYDNEY NSW 2000 Invention Title: 'EXTRACELLULAR MATRIX SIGNALLING MOLECULES' Details of Original Application No. 23296/97 dated 14 Mar 1997 The following statement is a full description of this invention, including the best method of performing it known to me/us:- File: 31426AUP00 -la- EXTRACELLULAR MATRIX SIGNALLING MOLECULES This application claims the benefit of the filing date of U.S. provisional patent application serial number 60/013,958, filed March 15, 1996.

FIELD OF THE INVENTION The present invention is directed to materials and methods involving extracellular matrix signalling molecules polypeptides involved in cellular responses to growth factors. More particularly, the invention is directed to Cyr61-, Fispl2-, and CTGF-related polynucleotides, polypeptides, compositions thereof, methods of purifying these polypeptides, and methods of using these polypeptides.

BACKGROUND OF THE INVENTION Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

S* The growth of mammalian cells is tightly regulated by polypeptide growth factors. In the adult animal, most cells are metabolically active but are quiescent with regard to cell division. Under certain conditions, these cells can be stimulated to reenter the cell cycle and divide. As quiescent cells reenter the active growth and division 20 phases of the cell cycle, a number of specific genes, the immediate early genes, are rapidly activated. Reentry to the active cell cycle is by necessity tightly regulated, since a breakdown of this control can result in uncontrolled growth, frequently recognized as cancer. Controlled reentry of particular cells into the growth phase is essential for such biological processes as angiogenesis blood vessel growth and repair), chondrogenesis skeletal development and prosthesis integration), oncogenesis lbcancer cell metastasis and tumor neovascularization), and other growth-requiring processes.

Angiogenesis, the formation of new blood vessels from the endothelial cells of preexisting blood vessels, is a complex process which involves a changing profile of endothelial cell gene expression, associated with cell migration, proliferation, and differentiation. Angiogenesis begins with localized breakdown of the basement membrane of the parent vessel. In vivo, ease o• *1 basement membranes (primarily composed of laminin, collagen type IV, nidogen/entactin, and proteoglycan) support the endothelial cells and provide a barrier separating these cells from the underlying stroma. The basement membrane also affects a variety of biological activities including cell adhesion, migration, and growth during development and differentiation.

Following breakdown of the basement membrane, endothelial cells migrate away from the parent vessel into the interstitial extracellular matrix (ECM), at least partially due to chemoattractant gradients. The migrating endothelial cells form a capillary sprout, which elongates. This 10 elongation is the result of migration and proliferation of cells in the sprout.

Cells located in the leading capillary tip migrate toward the angiogenic S: stimulus, but neither synthesize DNA nor divide. Meanwhile, behind these leading tip cells, other endothelial cells undergo rapid proliferation to ensure an adequate supply of endothelial cells for formation of the new vessel.

Capillary sprouts then branch at their tips, the branches anastomose or join with one another to form a lumen, the basement membrane is reconstituted, and a vascular connection is established leading to blood flow.

Alterations in at least three endothelial cell functions occur during- angiogenesis: 1) modulations of interactions with the ECM, which require alterations of cell-matrix contacts and the production of matrixdegrading proteolytic enzymes; 2) an initial increase and subsequent decrease in endothelial cell migration, effecting cell translocation towards an angiogenic stimulus; and 3) a transient increase in cell proliferation, providing cells for the growing and elongating vessel, with a subsequent return to the quiescent cell state once the vessel is formed. These three functions are realized by adhesive, chemotactic, and mitogenic interactions or responses, respectively.

Therefore, control of angiogenesis requires intervention in three distinct cellular activities: 1) cell adhesion, 2) cell migration, and 3) cell proliferation.

Another biological process involving a similar complex array of cellular activities is chondrogenesis.

-2- Chondrogenesis is the cellular process responsible for skeletal organization, including the development of bone and cartilage.

Chondrogenesis, like angiogenesis, involves the controlled reentry of quiescent cells into the growth phase of the cell cycle. The growth phase transition is associated with altered cell adhesion characteristics, changed patterns of cell migration, and transiently increased cell proliferation. Chondrogenesis involves the initial development of chondrogenic capacity the protodifferentiated state) by primitive undifferentiated mesenchyme cells. This stage involves the production of chondrocyte-specific markers without the 10 ability to produce a typical cartilage ECM. Subsequently, the cells develop the capacity to produce a cartilage-specific ECM as they differentiate into chondrocytes. Langille, Microscop. Res. Tech. 28:455-469 (1994).

Chondrocyte migration, adhesion, and proliferation then contribute to the development of bony, and cartilaginous, skeleton. Abnormal elaboration of the programmed development of cells participating in the process of chondrogenesis results in skeletal defects presenting problems that range from cosmetic concerns to life-threatening disorders.

Like angiogenesis and chondrogenesis, oncogenesis is characterized by changes in cell adhesion, migration, and proliferation.

Metastasizing cancer cells exhibit altered adhesion and migration properties.

Establishment of tumorous masses requires increased cell proliferation and the elaboration of the cellular properties characteristic of angiogenesis during the neovascularization of tumors.

Abnormal progression of angiogenesis or chondrogenesis, as well as mere progression of oncogenesis, substantially impairs the quality of life for afflicted individuals and adds to modern health care costs. The features common to these complex biological processes, comprising altered cell adhesion, migration, and proliferation, suggest that agents capable of influencing all three of these cellular activities would be effective in screening for, and modulating, the aforementioned complex biological processes.

-3- Although the art is aware. of agents that influence individual. cellular activities, integrins and selectins (cell adhesion), chemokines (cell migration), and a variety of growth factors or cytokines (cell proliferation), until recently no agent has been identified that exerts an influence over all three cellular activities in humans.

Murine Cyr61 (CYsteine-Rich protein) is a protein expressed in actively growing and dividing cells that may influence each of these three cellular activities. RNase protection analyses have shown that the gene encoding murine Cyr6i, murine cyr61, is transcribed in the developing mouse 10 embryo. O'Brien er al., Cell Growth Diff. 3:645-654 (1992). In situ hybridization analysis showed that expression of cyr61 during mouse einbryogenesis is closely correlated with the differentiation of mesenchymal cells, derived from ectoderm and mesoderm, into chondrocytes. In addition, cyr61 is expressed in the vessel walls of the developing circulatory system.

These observations indicate that murine cyr61 is expressed during cell proliferation and differentiation, which are characteristics of expression of genes involved in regulatory cascades that control the cell growth cycle.

Further characterization of the Cyr61 polypeptide has been hampered by an inability to purify useful quantities of the protein. Efforts to purify Cyr61 in quantity by overexpression from either eukaryotic or prokaryotic cells typically fail. Yang, University ofIllinois at Chicago, Ph.D.

Thesis (1993)._ One problem associated with attempting to obtain useful quantities of Cyr61 is the reduction in mammalian growth rates induced by overexpression of Cyr61. Another problem with Cyr61 purification is that the cysteine-rich polypeptide, when expressed in bacterial cells using recombinant DNA techniques, is often found in insoluble protein masses. Nevertheless, Cyr61 has been characterized as a polypeptide of 349 amino acids, containing 39 cysteine residues, a hydrophobic putative N-terninal signal sequence, and potential N-linked glycosylation sites (Asn,, and U.S. Patent No.

-4- *5,408,040 at column 3, lines 4.1-54, Grotendorst .et al., incorporated herein by reference (the '040 Patent).

Recently, proteins related to Cyr61 have been characterized.

For example, a human protein, Connective Tissue Growth Factor (CTGF), has been identified. (See '040 Patent). CTGF is expressed in actively growing cells such as fibroblasts and endothelial cells ('040 Patent, at column 5y lines 62-64), an expression pattern shared by Cyr61. In terms of function, CTGF has been described as a protein growth factor because its primary biological activity has been alleged to be its mitogenicity ('040 Patent, at column 2, lines 10 25-27 and 53-55). In addition, CTGF reportedly exhibits chemotactic activity.

'040 Patent, at column 2, lines 56-59. In terms of structure, the polynucleotide sequence encoding CTGF, and the amino acid sequence of CTGF, have been published. '040 Patent, SEQ ID NO:7 and SEQ ID NO:8, respectively.

Another apparently related protein is the mouse protein Fispl2 (FIbroblast Secreted Protein). Fispl2 has been subjected to amino acid sequence analysis, revealing a primary structure that is rich in cysteines.

Rvseck et al., Cell Growth Diff 2:225-233 (1991), incorporated herein by reference. The protein also possesses a hydrophobic N-terminal sequence suggestive of the signal sequence characteristic of secreted proteins.

Sequence analyses involving Cyr61, Fispl2, CTGF, and other proteins, have contributed to the identification of a family of cysteine-rich secreted proteins. Members of the family share similar primary structures encoded by genes exhibiting similar sequences. Each of the proteins in this emerging family is further characterized by the presence of a hydrophobic Ntenninal signal sequence and 38 cysteine residues in the secreted forms of the proteins. Members of the family identified to date include the aforementioned Cyr61 (human and mouse), Fispl2 (mouse), and CTGF (the human ortholog of Fispl2), as well as CEF10 (chicken), and Nov (avian).

One of several applications for a purified protein able to affect.

cell adhesion, migration, and proliferation properties involves the development of stable, long term ex vivo hematopoietic stem cell cultures. Patients subjected to high-dose chemotherapy have suppressed hematopoiesis; expansion of stem cells, their maturation into various hematopoietic lineages, and mobilization of mature cells into circulating blood routinely take-many weeks to complete. For such patients, and others who need hematopoietic cell transplantation, introduction into those patients of autologous stem cells that have been manipulated and expanded in culture is advantageous. Such 10 hematopoietic stem cells (HSC) express the CD34 stem cell antigen, but do not express lineage commitment antigens. These cells can eventually give rise to all blood cell lineages erythrocytes, lymphocytes, and myelocytes).

Hematopoietic progenitor cells that can initiate and sustain long term cultures long tenn culture system-initiating cells or LTC-IC) 15 represent a primitive population of stem cells. The frequency of LTC-IC has been estimated at only 1-2 per 104 cells in normal human marrow and only about 1 per 50-100 cells in a highly purified CD34' subpopulation. Thus, it would be useful to have methods and systems for long term cell culture that maintain and expand primitive, pluripotent human HSC to be used for 20 repopulation of the hematopoietic system in vivo.

Cell culture models of hematopoiesis have revealed a multitude of cytokines that appear to play a role in the hematopoietic process, including various colony stimulating factors, interleukins, stem cell factor, and the c-kit ligand. However, in ex vivo cultures, different combinations of these cytokines favor expansion of different sets of committed progenitors. For example, a factor in cord blood plasma enhanced expansion of granulocyteerythroid-macrophage-megakaryocyte colony forming unit (CFU-GEMM) progenitors, but expansion in these cultures favored the more mature subsets of cells. -Therefore, it has been difficult to establish a culture system that mimics in vivo hematopoiesis.

6 An HSC culture system should maintain and expand a large.

number of multi- or pluripotent stem cells capable of both long term repopulation and eventual lineage commitment under appropriate induction.

However, in most ex vivo culture systems, the fraction of the cell population comprised of LTC-IC decreases steadily with continued culturing, often declining to 20% of their initial level after several weeks, as the culture becomes populated by more mature subsets of hematopoietic progenitor cells that are no longer pluripotent. Moreover, the proliferative capacity exhibited by individual LTC-IC may vary extensively. Thus, a need exists in the art for 10 HSC culture systems comprising biological agents that maintain or promote the pluripotent potential of cells such as LTC-IC cells. In addition to a role in developing ex vivo HSC cultures, biological agents affecting cell adhesion, migration, and proliferation are useful in a variety of other contexts.

Proteins that potentiate the activity of mitogens but have no mitogenic activity themselves may play important roles as signalling molecules in such processes as hematopoiesis. Moreover, these signalling proteins could also serve as probes in the search for additional mitogens, many of which have not been identified or characterized. Several biological factors have been shown to potentiate the mitogenic activity of other factors, without being 20 mitogenic themselves. Some of these potentiators are associated with the cell surface and/or extracellular matrix. Included in this group are a secreted basic Fibroblast Growth Factor-binding protein (bFGF-binding protein), the basal lamina protein perlecan, and the Human Immunodeficiency Virus-1 TAT protein, each protein being able to promote bFGF-induced cell proliferation and angiogenesis. Also included in this group of mitogen potentiators are thrombospondin, capable of activating a latent form of Transforming Growth Factor-B. and an unidentified secreted growth-potentiating factor from vascular smooth muscle cells (Nakano er al., J. Biol. Chem. 270:5702-5705 [1995]), the latter factor being required for efficient activation of Epidermal Growth Factor- or thrombin-induced DNA synthesis. Further, the B cell stimulatory -7factor- /-interleukin-4, a T cell product with no demonstrable mitogenic activity, is able to 1) enhance the proliferative response of granulocytemacrophage progenitors to granulocyte-colony stimulating factor, 2) enhance the proliferative response of erythroid progenitors to erythropoietin, and 3) together with erythropoietin, induce colony formation by multipotent progenitor cells. Similarly, interleukin-7 enhanced stem cell factor-induced colony formation by primitive murine bone marrow progenitors, although interleukin-7 had no proliferative effect by itself. In addition, lymphocyte growth enhancing factor (LGEF) was found to enhance mitogen-stimulated 10 human peripheral blood lymphocyte (PBL) or purified T cell proliferation in a dose-dependent fashion. LGEF alone did not stimulate PBL or T cell proliferation.

a Therefore, a need continues to exist for biological agents capable of exerting a concerted and coordinated influence on one or more of 15 the particularized functions collectively characterizing such complex biological processes as angiogenesis, chondrogenesis, and oncogenesis. In addition, a need persists in the art for agents contributing to the reproduction of these in vivo processes in an ex vivo environment, the development of RSC cultures. Further, there continues to be a need for tools to search for the S 20 remaining biological components of these complex processes, mitogen probes, the absence of which impedes efforts to advantageously modulate and thereby control such processes.

-8- -9- SUMMARY OF THE INVENTION The present invention provides extracellular matrix (ECM) signalling moleculerelated materials and methods. In particular, the present invention is directed to polynucleotides encoding ECM signalling molecules and fragments or analogs thereof, ECM signalling molecule-related polypeptides and fragments, analogs, and derivatives thereof, methods of producing ECM signalling molecules, and methods of using ECM signalling molecules.

Accordingly, a first aspect of the invention provides an antibody that specifically binds to a polypeptide having the amino acid sequence set forth in SEQ ID NO:4, and analogs and/or derivatives of said polypeptide.

According to a second aspect, the invention provides a recombinant antibody that specifically binds to a polypeptide having the amino acid sequence set forth in SEQ ID NO: 4, and analogs and/or derivatives of said polypeptide.

A further aspect of the present invention relates to a purified and isolated polypeptide comprising an ECM signalling molecule. The polypeptides according to the invention retain at least one biological activity of an ECM signalling molecule, such as the ability to stimulate cell adhesion, cell migration, or cell proliferation; the ability to modulate angiogenesis, chondrogenesis, or oncogenesis; immunogenicity or the ability to elicit an immune response; and the ability to bind to polypeptides having specific binding sites for ECM signalling molecules, including antibodies and integrins. The polypeptides may be native or recombinant molecules. Further, the invention comprehends full-length ECM signalling molecules, and fragments thereof. In addition, the polypeptides of the invention may be underivatized, or derivatized in conformity with a native or non-native derivatization pattern. The invention further extends to 25 polypeptides having a native or naturally occurring amino acid sequence, and variants -9apolypeptides having different amino acid sequences), analogs polypeptides having a non-standard amino acid or other structural variation from the conventional set of amino acids) and homologs polypeptides sharing a common evolutionary ancestor with another polypeptide) thereof. Polypeptides that are covalently linked to other compounds, such as polyethylene glycol, or other proteins or peptides, i.e. fusion proteins, are contemplated by the invention.

Exemplary .ECM signalling molecules include mammalian Cyr61, Fispl2, and CTGF polypeptides. Beyond ECM signalling molecules, the invention includes polypeptides that specifically bind an ECM signalling molecule of the invention, such as the aforementioned antibody products. A wide variety of antibody products fall within the scope of the invention, including polyclonal and monoclonal antibodies, antibody fragments, chimeric antibodies, CDR-grafted antibodies, "humanized" antibodies, and other antibody forms known in the art. Other molecules such as peptides, carbohydrates or lipids designed to bind to an active site of the ECM 10 molecules thereby inhibiting their activities are also contemplated by the invention. However molecules such as peptides that enhance or potentiate the activities of ECM molecule are also within the scope of the invention. The invention further extends to a pharmaceutical composition comprising a biologically effective amount of a polypeptide and a pharmaceutically 15 acceptable adjuvant, diluent or carrier, according to the invention. A "biologically effective amount" of the biomaterial is an amount that is sufficient to result in a detectable response in the biological sample when compared to a control lacking the biomaterial.

Another aspect of the invention relates to a purified and isolated 20 polynucleotide comprising a sequence that encodes a polypeptide of the invention. A polynucleotide according to the invention may be DNA or RNA, single- or double-stranded, and may be may purified and isolated from a native source, or produced using synthetic or recombinant techniques known in the art. The invention also extends to polynucleotides encoding fragments, analogs polynucleotides having a non-standard nucleotide), homologs polynucleotides having a common evolutionary ancestor with another polynucleotide). variants polynucleotides differing in nucleotide sequence), and derivatives polynucleotides differing in a structural manner that does not involve the primary nucleotide sequence) of ECM molecules. Vectors comprising a polynucleotide according to the invention 10 are also contemplated. In addition, the' invention comprehends host cells transformed or transfected with a polynucleotide or vector of the invention.

Other aspects of the invention relate to methods -for maiking-or using the polypeptides and/or polynucleotides of the invention. A method for making a polypeptide according to the invention comprises expressing a polynucleotide encoding a polypeptide according to the present invention in a suitable host cell and purifying the polypeptide. Other methods for making a polypeptide of the invention use techniques that are known in the art, such as the isolation and purification of native polypeptides or the use of synthetic 10 techniques for polypeptide production. In particular, a method of purifying an ECM signalling molecule such as human Cyr61 comprises the steps of identifying a source containing human Cyr61, exposing the source to a human Cyr61-specific biomolecule that binds Cyr61 such as an anti-human Cyr61 *i antibody, and eluting the human Cyr61 from the antibody or other 15 biomolecule, thereby purifying the human Cyr61.

Another aspect of the invention is a method of screening for a modulator of angiogenesis comprising the steps of: contacting a first biological sample capable of undergoing angiogenesis with a biologically effective angiogenically effective) amount of an ECM signalling 20 molecule-related biomaterial and a suspected modulator (inhibitor or potentiator); separately contacting a second biological sample with a biologically effective amount of an ECM signalling molecule-related biomaterial, thereby providing a control; measuring the level of angiogenesis resulting from step and from step and comparing the levels of angiogenesis measured in step whereby a modulator of angiogenesis is identified by its ability to alter the level of angiogenesis when compared to the control of step The modulator may be either a potentiator or inhibitor of angiogenesis and the ECM signalling moleculerelated biomaterial includes, but is not limited to, Cyr61, and fragments.

variants, homologs, analogs, derivatives, and antibodies thereof.

11 The invention also extends to a method of screening for a modulator of angiogenesis comprising the steps of: preparing a first implant comprising Cyr61 and a second implant comprising Cyr61 and a.

suspected modulator of Cyr61 angiogenesis; implanting the first implant in a first cornea of a-test animal and the second implant in a second cornea of the test animal; measuring the development of blood vessels in the first and second corneas; and comparing the levels of blood vessel development measured in step whereby a modulator of angiogenesis is identified by its ability to alter the level of blood vessel development in the first cornea when i* 10 compared to the blood vessel development in the second cornea.

Another aspect of the invention relates to a method of screening for a modulator of chondrogenesis comprising the steps of: contacting a first biological sample capable of undergoing chondrogenesis with a biologically effective chondrogenically effective) amount of an ECM 15 signalling molecule-related biomaterial and a suspected modulator; (b) separately contacting a second biological sample capable of undergoing chondrogenesis with a biologically effective amount of an ECM signalling molecule-related biomaterial, thereby providing a control; measuring the level of chondrogenesis resulting from step and from step and (d) 20 comparing the levels of chondrogenesis measured in step whereby a modulator of chondrogenesis is identified by its ability to alter the level of chondrogenesis when compared to the,control of step The modulator may be either a promoter or an inhibitor of chondrogenesis; the ECM signalling molecules include those defined above and compounds such as mannose-6phosphate, heparin, and tenascin.

The invention also relates to an in vitro method of screening for a modulator of oncogenesis comprising the steps of: inducing a first tumor and a second tumor; administering a biologically effective amount of an ECM signalling molecule-related biomaterial and a suspected modulator to the first tumor: separately administering a biologically effective amount of an 12 ECM signalling molecule-related biomaterial to the second tumor, thereby providing a control; measuring the level of oncogenesis resulting from step and from step and comparing the levels of oicogenesis measured in step whereby a modulator of oncogenesis is identified by its ability to alter the level of oncogenesis when compared to the control of step Modulators of oncogenesis contemplated by the invention include inhibitors of oncogenesis. Tumors may be induced by a variety of techniques including, but not limited to, the administration of chemicals, carcinogens, and the implantation of cancer cells. A related aspect of the invention is a method for 10 treating a solid tumor comprising the step of delivering a therapeutically effective amount of a Cyr61 inhibitor to an individual, thereby inhibiting the neovascularization of the tumor. Inhibitors include, but are not limited to, inhibitor peptides such as peptides having the "RGD" motif, and cytotoxins, which may be free or attached to molecules such as Cyr61.

S 15 Yet another aspect of the invention is directed to a method of screening for a modulator of cell adhesion comprising the steps of: (a) preparing a surface compatible with cell adherence; separately placing first and second biological samples capable of undergoing cell adhesion on the surface; contacting a first biological sample with a suspected modulator 20 and a biologically effective amount of an ECM signalling molecule-related biomaterial selected from the group consisting of a human Cyr61, a human Cyr61 fragment, a human Cyr61 analog, and a human Cyr61 derivative; (d) separately contacting a second biological sample with a biologically effective amount of an ECM signalling molecule-related biomaterial selected from the group consisting of a human Cyr61, a human Cyr61 fragment, a'human Cyr61 analog, and a human Cyr61 derivative, thereby providing a control; (e) measuring the level of cell adhesion resulting from step and from step and comparing the levels of cell adhesion measured in step whereby a modulator of cell adhesion is identified by its ability to alter the level of cell adhesion when compared to the control of step 13 The invention also extends to a method of screening for a modulator of cell migration comprising the steps of: forming a gel matrix comprising Cyr61 and a suspected modulator of cell migration; preparing a control gel matrix comprising Cyr61; seeding endothelial cells capable of undergoing cell migration onto the gel matrix of step and the control gel matrix of step incubating the endothelial cells; measuring the levels of cell migration by inspecting the interior of the gel matrix and the control gel matrix for cells; comparing the levels of cell migration measured in step whereby a modulator of cell migration is identified by its ability to 1 0 alter the level of cell migration in the gel matrix when compared to the level of cell migration in the control gel matrix. The endothelial cells include, but are not limited to, human cells, human microvascular endothelial cells.

The matrix may be formed from gelling materials such as Matrigel, collagen, or fibrin, or combinations thereof.

15 Another aspect of the invention is directed to an in vitro method of screening for cell migration comprising the steps of: fonning a first gelatinized filter and a second gelatinized filter, each filter having two sides; contacting a first side of each the filter with endothelial cells, thereby :adhering the cells to each the filter; applying an ECM signalling molecule 20 and a suspected modulator of cell migration to a second side of the first gelatinized filter and an ECM signalling molecule to a second side of the second gelatinized filter; incubating each the filter; detecting cells on the second side of each the filter; and comparing the presence of cells on the second side of the first gelatinized filter with the presence of cells on the second side of the second gelatinized filter, whereby a modulator of cell migration is identified by its ability to alter the level of cell migration.

measured on the first gelatinized filter when compared to the cell migration measured on the second gelatinized filter. The endothelial cells are defined Sabove. The ECM signalling molecules extend to human Cyr61 and each of 14the filters may be placed in apparatus such .as a -Boyden chamber, including modified Boyden chambers.

The invention also embraces a in vi vo method of screening for a modulator of cell migration comprising the steps of: removing a first central portion of a first biocompatible sponge and a second central portion of a second biocompatible sponge; applying an ECM signalling molecule and a suspected modulator to the first central portion and an ECM signalling molecule to the second central portion; reassociating the first central portion with said first biocompatible sponge and said second central portion 10 with the second biocompatible sponge; attaching a first filter to a first side .of the first biocompatible sponge and a second filter to a second side of the first biocompatible sponge; attaching a third filter to a first side of the second biocompatible sponge and a fourth filter to a second side of the second biocompatible sponge; implanting each of the biocompatible sponges, each biocompatible sponge comprising the central portion and the filters, in a test animal; removing each the sponge following a period of incubation; (f) measuring the cells found within each of the biocompatible sponges; and (g) comparing the presence of cells in the first biocompatible sponge with the .presence of cells in the second biocompatible sponge, whereby a modulator 20 of cell migration is identified by its ability to alter the level of cell migration measured using the first biocompatible sponge when compared to the cell migration measured using the second biocompatible sponge. ECM signalling molecules include, but are not limited to, human Cyr61; the ECM signalling molecule may also be associated with Hydron. In addition, the in vivo method of screening for a modulator of cell migration may include the step of providing a radiolabel to the test animal and detecting the radiolabel in one or more of the sponges.

Another aspect of the invention relates to a. method for modulating hemostasis comprising the step of administering an ECM signalling molecule in a pharmaceutically acceptable adjuvant, diluent or 15 carrier. Also, the invention extends to a method of:inducing wound healing in a tissue comprising the step of contacting a wounded tissue with a biologically effective amount of an ECM signalling molecule, thereby promoting wound healing. The ECM signalling molecule may be provided in the form of an ECM signalling molecule polypeptide or an ECM signalling molecule nucleic acid, using a gene therapy technique. For example, the nucleic acid may comprise an expression control sequence operably linked to an ECM signalling molecule which is then introduced into the cells of a wounded tissue. The expression of the coding sequence is controlled, e.g., 10 by using a tissue-specific promoter such as the K14 promoter operative in skin tissue to effect the controlled induction of wound healing. The nucleic acid may include a vector such as a Herpesvirus, an Adenovinis, an Adenoassociated Virus, a Cytomegalovirus, a Baculovirus, a retrovirus, and a Vaccinia Virus. Suitable wounded tissues for treatment by this method 15 include, but are not limited to, skin tissue and lung epithelium. A related method comprises administering a biologically effective amount of an ECM signalling molecule, e.g. Cyr61, to an animal to promote organ regeneration.

The impaired organ may be the result of trauma, e.g. surgery, or disease.

Another method of the invention relates to improving the vascularization of grafts, skin grafts. Another method of the invention is directed to a process for promoting bone implantation, including bone grafts. The method for promoting bone implantation comprises the step of contacting a bone implant or receptive site with a biologically effective chondrogenically effective) amount of an ECM signalling molecule. The contacting step may he effected by applying the ECM signalling molecule to a biocompatible wrap such as a biodegradable gauze and contacting the wrap-with a bone implant, thereby promoting bone implantation. The bonie implants comprise natural bones and fragments thereof, as well as inanimate natural and synthetic materials that are biocompatible, such as prostheses. In addition to direct application of an ECM signalling molecule to a bone, prosthesis, or receptive 16site, the invention contemplates the-use .of matrix .materials for controlled release of the ECM signalling molecule, in addition to such application materials as gauzes.

Yet another aspect of the invention relates to a method of screening for a modulator of cell proliferation comprising the steps of: (a) contacting a first biological sample capable of undergoing cell proliferation with a suspected modulator and a biologically effective mitogenically effective) amount of an ECM signalling molecule-related biomaterial selected from the group consisting of a human Cyr61, a human Cyr61 fragment, a 10 human Cyr61 analog, and a human Cyr61 derivative; separately contacting a second biological sample capable of undergoing cell proliferation with a biologically effective amount of an ECM signalling molecule-related biomaterial selected from the group consisting of a human Cyr61, a human "i Cyr61 fragment, a human Cyr61 analog, and a human Cyr61 derivative, thereby providing a control; incubating the first and second biological samples; measuring the level of cell proliferation resulting from step .and comparing the levels of cell proliferation measured in step whereby a modulator of cell proliferation is identified by its ability to alter the level of cell adhesion when compared to the control of step 20 Also comprehended by the invention is a method for expanding a population of undifferentiated hematopoietic stem cells in culture, comprising the steps of: obtaining hematopoietic stem cells from a donor; and culturing said cells under suitable nutrient conditions in the presence of a biologically effective hematopoietically effective) amount of Cyr61.

Another method according to the invention is a method of screening for a mitogen comprising the steps of: plating cells capable of undergoing cell proliferation; contacting a first portion of the cells with a solution comprising Cyr61 and a suspected mitogen; contacting a second portion of the cells with a solution comprising Cyr61, thereby providing a control: incubating the cells; detecting the growth of the first portion 17of cells and the second portion of the cells; and comparing growth of the first and second portions of cells, whereby a mitogen is identified by its ability to induce greater growth in the first portion of cells when compared to the growth of the second portion of cells. The cells include, but are not limited to, endothelial cells and fibroblast cells. Further, the method may involve contacting the cells with a nucleic acid label, 3 H]-thymidine. and detecting the presence of the label in the cells. Another method relates to improving tissue grafting, comprising administering to an animal a quantity of Cyr61 effective in improving the rate of neovascularization of a graft.

10 Numerous additional aspects and advantages of the present invention will be apparent upon consideration of the following drawing and detailed description.

18 BRIEF DESCRIPTION OF THE DRAWING FIGURE 1 presents the comparative amino acid sequences of members of the cysteine-rich protein family of growth-regulating p~oteins.

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a 5( S S

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19 DETAILED DESCRIPTION OF THE INVENTION In the mouse, the Cyr61 protein has been found to influence cell adhesion, migration, and proliferation. The cyr61 gene, which encodes Cyr61, is an immediate-early gene that is transcriptionally activated by serum growth factors in mouse fibroblasts. Lau et al., EMBO J. 4:3145-3151 (1985.), incorporated herein by reference; Lau et al., Proc. Natl. Acad. Sci.

(USA) 84:1182-1186 (1987), incorporated herein by reference. The murine cyr61 cDNA coding sequence is set forth in SEQ ID NO:1. (The human cyr61 cDNA coding sequence is provided in SEQ ID NO:3). The amino acid 10 sequence of murine Cyr61 is set out in SEQ ID NO:2. (The human Cyr61 amino acid sequence is presented in SEQ ID NO:4). Cyr61 is a 41 kDa polypeptide exhibiting 39 cysteine residues, approximately 10% of the 379 amino acids constituting the unprocessed protein. Yang et al., Cell Growth Diff 2:351-357 (1991), incorporated herein by reference. Investigations 15 have revealed that murine Cyr61 binds heparin and is secreted. Yang et al.

Consistent with the observed -secretion of Cyr61 is the identification of an Neo* terminal signal sequence in nascent Cyr61, deduced from inspection of the n murine cyr61 cDNA sequence. Yang et al. Additionally, Cyr61 is not found in the. conditioned medium of cultured cells expressing cvr61, but is found 20 associated with the extracellular matrix (ECM) and the cell surface. Yang et al. Structurally similar cysteine-rich mammalian proteins have been characterized.

Fispl2, a cysteine-rich murine protein, exhibits structural similarity to Cyr61. The cDNA sequence encoding Fispl2 is set forth in SEQ ID NO:5: the amino acid sequence of Fispl2 is presented in SEQ ID NO:6.

Murine Fispl2, like Cyr61, influences cell adhesion, proliferation and migration. The human ortholog of Fispl2 is Connective Tissue Growth Factor (CTGF), a protein similar in structure and function to Cyr61. Fispl2, and CTGF. are distinguishable from Cyr61, however. For example, a greater proportion of secreted Fispl2 is found in the culture medium than is the case with Cyr61;-a correspondingly-lower proportion -of Fisp 12 is localized in the area of expressing cells (cell surface and nearby extracellular matrix) than is found with Cyr61. Additional similarities and distinctions among the proteins comprising the ECM signalling molecules of the invention will become apparent in the recitations hereinbelow.

The present invention has multiple aspects, illustrated by the following examples. Example 1 describes the cloning of polynucleotides encoding members of the cysteine-rich protein family of ECM signalling molecules; Example 2 describes sequence analyses; Example 3 describes RNA 10 analyses; Example 4 describes the production of transgenic animals; Example 5 describes the expression of Cyr61 polypeptides; Example 6 describes the expression of Fispl2 polypeptides; Example 7 sets out methods of polypeptide purification; Example 8 provides a characterization of the polypeptides of the invention; Example 9 discloses a heparin binding assay for the polypeptide 15 members of the cysteine-rich protein family; Example 10 is directed to receptors for the polypeptides; Example 11 describes anti-ECM signalling molecule antibodies; Example 12 is directed to inhibitory peptides; Example 13 describes cell adhesion and polypeptide-based methods for influencing the process of cell adhesion; Example 14 describes polypeptide-influenced migration of fibroblasts; Example 15 describes the migration of endothelial cells and in vitro assays for migration; Example 16 describes an in vitro assay for inhibitors of endothelial cell migration; Example 17 describes an in vivo assay for endothelial cell migration; Example 18 describes mitogen potentiation by the polypeptides of the invention, Example 19 describes an in vivo cornea assay for angiogenic factors and modulators; Example 20 is directed to methods for influencing blood clotting using the polypeptides of the invention; Example 21 discloses the use of the polypeptides for ex vivo hematopoietic stem cell cultures; Example 22 addresses organ regeneration: Example 23 describes chondrogenesis and the expression of extracellular matrix signalling molecules in mesenchyme cells: Example 24 describes the -21 promotion of cell adhesion in the process of chondrogenesis using the polypeptides of the invention; Example 25 describes chondrogenesis and the influence of the polypeptides of the invention on cell aggregation; Example 26 describes the promotion of cell proliferation by polypeptides of the invention in the process of chondrogenesis; Example 27 addresses methods for using the polypeptides of the invention to affect chondrogenesis; and Exampl'e 28 provides genetic approaches to the use of polynucleotides of the invention.

These examples are intended to be illustrative of the present invention and should not be construed to limit the scope of the invention.

*e Example 1 Polynucleotide Cloning A human cyr61 cDNA was isolated from a human placental cDNA library by probing with the murine cyr61 cDNA sequence using Stechniques that are standard in the art. See Sambrook et al., incorporated herein by reference. Isolation of the complete murine cyr61 cDNA from a BALB/c 3T3 (ATCC CRL-1658) cDNA library has been described. O'Brien et al., Mol. Cell. Biol. 10:3569-3577 (1990), incorporated herein by reference. The nucleotide and deduced amino acid sequences of murine cyr61 are available from the GenBank database under accession number M32490.

The nucleotide sequence of murine cyr61 is presented in SEQ ID NO: 1; the murine Cyr61 amino acid sequence is presented in SEQ ID NO:2.

The human cDNA library was constructed using Xgtll (Promega Corp., Madison, WI) as a vector which was transfected into E. coli and plated on LB agar. A murine cDNA expression construct cloned in pGEM-2 (O'Brien et al., [1990]), containing the entire murine cyr61 coding sequence [nucleotides 56-1560, using the numbering of O'Brien et al., (1990); see SEQ ID NO:1] was used as a probe. The mouse cDNA probe was radiolabeled by techniques standard in the art. Sambrook et al. Plaque 22 screenings using the mouse probe were performed using standard techniques.

Sambrook et al.

More particularly, agar plates containing the human cDNA library described above were exposed to nitrocellulose filters (BA85, 82 mm, Schleicher Schuell, Keene, NH) were placed on each plate. After plaque adsorption (approximately 20 minutes), the filters were removed and air'dried for approximately 30 minutes. Subsequently, each filter was sequentially submerged for 30-60 seconds in 0.2M NaOH, 1.5M NaCI (100 ml); 2X SSC, 0.4M Tris-HC1, pH 7.4 (100 ml); and 0.2X SSC (100 ml). Filters were then 10 dried at room temperature for approximately 1 hour and subjected to under vacuum for 2 hours. Filters were probed with radiolabeled murine Scyri61 cDNA.

Alternatively, human cyr6l cDNA clones were identified with probes generated by RT-PCR. In particular, the probe for screening the 15 human placental cDNA library was a PCR fragment generated with degenerate primers by RT-PCR of total RNA from logarithmically growing WI38 cells.

The primers were derived from the sequences corresponding to the most conserved region of the open reading frame of the mouse cyr61 cDNA. One primer, designated H61-5 20 G(TC)AA(GA)GT(GC)TG-3'], contains a degenerate sequence which, with the exception of the "GGGAATTC" sequence at the 5' end which was used to introduce an EcoRI site, is derived from nucleotides 327-346 (sense strand) of the mouse cyr61 sequence set forth in SEQ ID NO: 1. The degeneracies appear in positions corresponding to the third position of codons in SEQ ID NO: 1. The second primer used for PCR amplification of a human cyr61 sequence was designated H61-3 A(GA)TT(GA)CA-3'], which, with the exception of the 5' sequence "CCGGATCC" used to introduce a BamHI site, corresponds to the anti-sense strand complementary to nucleotides 1236-1250 of the mouse cvr61 sequence set forth in SEQ ID NO: 1. The degeneracies occur in positions 23 complementary to. the third positions of codons in mouse cyr6 as set forth in SEQ ID NO: 1. The amplified cyr61 cDNA was cloned into the pBlueScript SK+ vector (Stratagene, La Jolla, CA) and sequienced with a Sequenase II kit Biochemicals, Cleveland, OH).

Serial screenings of the human placental cDNA library led to the isolation of a clone containing a human cyr61 cDNA. The human yevr61 cDNA is approximately 1,500 bp in length. The human cDNA is contained on an EcoRI fragment cloned into the EcoRI site in pGEM-2. As shown in SEQ ID NO:3, the human cDNA sequence includes the entire coding region 10 for human Cyr61, along with 120 bp of 5' flanking sequence, and about 150 bp of 3' flanking sequence.

The polynucleotides of the invention may be wholly or partially synthetic, DNA or RNA, and single- or double-stranded. Because polynucleotides of the invention encode ECM signalling molecule polypeptides 15 which may be fragments of an ECM signalling molecule protein, the polynucleotides may encode a partial sequence of an ECM signalling molecule. Polynucleotide sequences of the invention are useful for the production of ECM signalling molecules by recombinant methods and as hybridization probes for polynucleotides encoding ECM signalling molecules.

S 20 DNA polynucleotides according to the invention include genomic DNAs, cDNAs, and oligonucleotides comprising a coding sequence of an ECM signalling molecule, or a fragment or analog of an ECM signalling molecule, as described above, that retains at least one of the biological activities of an ECM signalling molecule such as the ability to promote cell adhesion, cell migration, or cell proliferation in such biological processes as angiogenesis, chondrogenesis, and oncogenesis, or the ability to elicit an antibody recognizing an ECM signalling molecule.

Other polynucleotides according to the invention differ in sequence from sequences contained within native ECM signalling molecule polynucleotides by the addition, deletion, insertion, or substitution of -24 nucleotides) provided the polynucleotides encode a protein that retains at least one of the biological activities of an ECM signalling molecule. A polynucleotide sequence of the invention may differ from a-native ECM signalling molecule polynucleotide sequence by silent mutations that do not 5 alter the sequence of amino acids encoded therein. Additionally, polynucleotides of the invention may specify an ECM signalling molecule that differs in amino acid sequence from native ECM signalling molecule sequences or subsequences, as described above. For example, polynucleotides encoding polypeptides that differ in amino acid sequence from native ECM 10 signalling molecules by conservative replacement of one or more amino acid residues, are contemplated by the invention. The invention also extends to polynucleotides that hybridize under standard stringent conditions to polynucleotides encoding an ECM signalling molecule of the invention, or that would hybridize but for the degeneracy of the genetic code. Exemplary 15 stringent hybridization conditions involve hybridization at 42 0 C in formamide, 5X SSC, 20 mM Na*PO 4 pH 6.8 and washing in 0.2X SSC at 0 C. It is understood by those of skill in the art that variation in these conditions occurs based on the length and GC nucleotide content of the sequences to be hybridized. Formulas standard in the art are appropriate for 20 determining exact hybridization conditions. See Sambrook et al., Molecular Cloning: A Laboratory Manual (Second ed., Cold Spring Harbor Laboratory Press 1989) 9.47-9.51.

ECM signalling molecule polynucleotides comprising RNA are also within the scope of the present invention. A preferred RNA polynucleotide according to the invention is an mRNA of human cyr6l. Other RNA polynucleotides of the invention include RNAs that differ from a native ECM signalling molecule mRNA by the insertion, deletion, addition, or substitution of nucleotides (see above), with the proviso that they encode a polypeptide retaining a biological activity associated with an ECM signalling molecule. Still other RNAs of the invention include anti-sense RNAs 25 RNAs comprising an RNA sequence that is complementary to an ECM signalling molecule mRNA).

Accordingly, in another embodiment a set of DNA fragments collectively spanning the human cyr61 cDNA were cloned in pGEM-2 and M13 derivatives using methods well known in the art to facilitate nucleotide sequence analyses. The pGEM-2 clones provided substrates for the enzymatic generation of serial deletions using techniques known in the art. This collection of clones, collectively containing a series of DNA fragments spanning various parts of the cyr61 cDNA coding region, are useful in the 10 methods of the invention. The resulting series of nested pGEM-2 clones, in turn, provided substrates for nucleotide sequence analyses using the enzymatic chain tenninating technique. The fragments are also useful as nucleic acid probes and for preparing Cyr61 deletion or truncation analogs. For example, the cyr61 cDNA clones may be used to isolate cyr61 clones from human 15 genomic libraries that are commercially available. (Clontech Laboratories, Inc., Palo Alto, CA). Genomic clones, in turn, may be used to map the cyr61 locus in the human genome, a locus that may be associated with a known disease locus.

Other embodiments involve the polynucleotides of the invention 20 contained in a variety of vectors, including plasmid, viral prokaryotic and eukaryotic viral vectors derived from Lambda phage, Herpesviruses, Adenovirus, Adeno-associated viruses, Cytomegalovirus, Vaccinia Virus, the M 13-fl -fd family of viruses, retroviruses, Baculovirus, and others), phagemid, cosmid, and YAC Yeast Artificial Chromosome) vectors.

Yet other emnbodiments involve the polynucleotides of the invention contained within heterologous polynucleotide environments.

Polynucleotides of the invention have been inserted into heterologous genomes, thereby creating transgenes, and transgenic animals, according to the invention. In particular, two types of gene fusions containing partial murine cvi61 gene sequences have been used to generate transgenic mice.

-26- (See below). One type of.fused gene recombined the coding sequence of cyr61 with one of three different promoters: 1) the K14 keratin promoter, 2) the 0-actin promoter, or 3) the phosphoglycerokinase promoter. Adra et al., Gene 60:65-74 (1987). These fusion constructs were generated using standard techniques, as described below in the context of a phosphoglycerokinase promoter (pgk-l)-cyr61 fusion. An XhoI-ScaI genomic DNA fragment containing the entire cyr61 coding region and all introns, but lacking the transcription initiation site and polyadenylation signal, was cloned into plasmid pgk/3-gal, replacing the lacZ coding sequence. The resulting construct placed 10 cyr61 under the control of the strong pgk-1 promoter which is active in all cells.

The second type of gene fusion recombined the cvr61 expression control sequences promoter) with the E. coli 0-galactosidase coding sequence. The cyr61-lacZ fusion gene was constructed using the following approach. A DNA fragment spanning nucleotides -2065 to relative to the transcription initiation nucleotide was used to replace the pgk-1 .promoter (Adra et al., Gene 60:65-74 [1987]) in plasmid pgk/0-gal by bluntend cloning. In addition, the polyadenylation signal from the bovine growth hormone gene was cloned into the plasmid containing the fusion gene. The resulting construct, plasmid 2/lacZ, has the E. coli lacZ gene under the transcriptional control of a 2 kb DNA fragment containing the cyr61 promoter. The related plasmid 1.4/lacZ was derived from plasmid 2lacZ by removing about 600 bp of cyr61 DNA found upstream of an AflII site. Also, plasmid 2M/lacZ resembles plasmid 2/lacZ, except for a C-to-T transition in the CArG Box, created by PCR. These constructs were excised from the vectors by NotI digestion, purified using GeneClean [BiolOl, Inc., La Jolla, CA). and used to generate transgenic mice (see below).

A cDNA fragment encoding mouse fispl2 has also been cloned using standard techniques. Ryseck et al., Cell Growth Diff. 2.225-233 (1991). incorporated herein by reference. The cloning was accomplished by 27 ligating an Xholl fragment containing the fispl2 cDNA coding region into BamHI-cleaved pBlueBacII, a baculovirus expression vector (Invitrogen Corp., San Diego TCA)_ -Recoibinantt baculovinis cloies were obtained as described in Summers et al., X Ag. Exp. Sta., Bulletin 1555 (1987).

The human ortholog offispl2,--the gene encoding CTGF, -was cloned by screening a fusion cDNA library with anti-Platelet-Derived Growth Factor (anti-PDGF) antibodies, as described in U.S. Patent No. 5,408,040, column 12, line 16, to column 13, line 29, incorporated herein by reference.

The screening strategy exploited the immunological cross-reactivity of CTGF 10 and PDGF.

The cloned copies of the cyr61,fispl2, and ctgfcDNAs provide a ready source for polynucleotide probes to facilitate the isolation of genomic coding regions, as well as allelic variants of the genomic DNAs or cDNAs.

In addition, the existing cDNA clones, or clones isolated by probing as 15 described above, may be used to generate transgenic organisms. For example, transgenic mice harboring cyr61 have been generated using standard techniques, as described in the next Example.

A clone, hCyr6lcDNA, containing the human cyr61 cDNA sequence set forth in SEQ ID NO: 3, and a bacterial strain transformed with S 20 that clone, Escherichia coli DH5a (hCyr6lcDNA), were deposited with the American Type Culture Collection 12301 Parklawn Drive, Rockville, MD 20852 USA, on March 14, 1997.

Example 2 Sequence Analyses The nucleotide sequence of murine cyr61 has been described, O'Brien et al. (1990); Latinkic et al., Nucl. Acids Res. 19:3261-3267 (1991), and is set out herein as SEQ ID NO: 1.

The deduced amino acid sequence of murine Cyr61 has been reported. O'Brien er al. (1990), and is set forth in SEQ ID NO:2.

28- The nucleotide sequence of the human cyr61 cDNA was determined using the method of Sanger, as described in Sambrook et al.

Sequencing templates were generated by constructing a series of nested deletions from a pGEM-2 human cyr61 cDNA clone, as described in Example 1 above. The human cyr61 cDNA sequence is set forth in SEQ ID NO:3.

The amino acid sequence of human Cyr61 was deduced from the human.cyr61 cDNA sequence and is set forth in SEQ ID NO:4.

A comparison of the mouse and human Cyr61 sequences, presented in SEQ ID NO:2 and SEQ ID NO:4, respectively, reveals 91% 10 similarity. Both sequences exhibit an N-terminal signal sequence indicative of a processed and secreted protein; both proteins also contain 38 cysteine residues, distributed throughout both proteins but notably absent from the central regions of both murine and human Cyr61. Notably, the region of greatest sequence divergence between the mouse and human Cyr61 coding regions is this central region free of cysteine residues. However, the untranslated regions of the mouse and human cyr61 cDNAs are even more divergent (67% similarity). In contrast, the 3' untranslated regions are the most similar regions (91 similarity). In overall length, the encoded murine Cyr61 has 379 amino acids; human Cyr61 has 381 amino acids.

S 20 A fispl2 cDNA sequence has also been determined and is set out in SEQ ID NO:5. The amino acid sequence of Fispl2 has been deduced from the fispl2 cDNA sequence and is set forth in SEQ ID NO:6. A comparison of the amino acid sequences of murine Cyr61 and Fispl2 reveals that the two proteins are 65% identical. The structural similarity of Cyr61 and Fispl2 is consistent with the similar functional properties of the two -proteins, described below.

A partial cDNA sequence of CTGF, containing the complete CTGF coding region, has also been determined. The CTGF cDNA sequence was obtained using M13 clones as templates for enzymatic sequencing reactions, as described. '040 Patent, at column 12, line 68 to column 13, line 29 14. Additional cloning coupled with double-stranded enzymatic sequencing reactions, elucidated the entire sequence of the cDNA encoding CTGF. U.S.

Patent No75,4G0804,07coT6umn 14, line44, to column 15, line 8, incorporated herein by reference. The nucleotide sequence of the cDNA encoding CTGF is presented herein -in -SEQ -ID NO:7. -The-deduced amino acid sequence of the cDNA encoding CTGF is presented in SEQ ID NO:8.

Example 3 RNA Analyses Polynucleotide probes are useful diagnostic tools for angiogenic, 10 and other, disorders correlated with Cyr61 expression because properly designed probes can reveal the location, and level, of cyr61 gene expression at the transcriptional level. The expression of cyr61, in turn, indicates whether or not genes controlling the process of angiogenesis are being expressed at typical, or expected, levels.

Using these tools, the mouse cyr61 mRNA expression pattern was determined using an RNase protection technique. O'Brien et al., (1992).

In particular, a 289 nucleotide antisense riboprobe was used that would protect 246 nucleotides of the murine cyr61 mRNA (nucleotides 67 to 313 using the .numbering of O'Brien et al.) The assays showed levels of cyr61 mRNA in PSA-1 cells (10 lg of total RNA) from either the undifferentiated state or stages 1, 2, and 3 of differentiation (PSA-1 cells undergo three stages of cellular differentiation corresponding to mouse embryonic cells of the following gestational ages, in days: 4.5-6.5 [PSA-1 stage 6.5-8.5 [PSA-1 stage 8.5-10.5 [PSA-1 stage A comparison of the protection of whole embryonic and placental total RNAs (20 pg each) showed that cyr61 is expressed in embryonic tissues at times that are coincident with the processes of cell differentiation and proliferation.

Expression characteristics of human cyr61 were determined by Northern analyses, using techniques that are standard in the art. Sambrook et al. RNA was isolated from the human diploid fibroblastic cell line WI38 (ATCC CCL-75). In addition, RNA was isolated from rat cells (REF52), hamster cells (CHO),-and-monkey cells (BSC40). -Each of the cell lines was grown to confluence in MEM-10 (Eagle's Minimal Essential Medium with Earle's salts [GIBCO-BRL, Inc.], 2 mM glutamine, and 10% fetal calf serum [fcs]) and maintained in MEM-0.5 (a 0.5% serum medium) for two 'days.

Cultures were then stimulated with 20% fcs, in the presence or absence of cycloheximide, by techniques known in the art. Lau et al. (1985; 1987). Ten microgram aliquots of RNA isolated from these cell lines were then 10 fractionated by fonnaldehyde-agarose gel electrophoresis, transferred and immobilized on nitrocellulose filters, and exposed to a full-length radiolabeled murine cyr61 cDNA probe under hybridization conditions of high stringency. Human cyr61 RNA expression was similar to murine cyr61 expression. Both mouse and human cyr61 expression yielded approximately 15 2 kilobase RNAs. Additionally, both mouse and human expression of Cyr61 were stimulated by serum and were resistant to cycloheximide.

The distribution of human cyr61 mRNA was also examined using multiple tissue Northern blots (Clontech). The blots were hybridized in an ExpressHyb Solution (Clontech) according to the manufacturer's instructions. The results showed that cyr61 mRNA is abundant in the human heart, lung, pancreas, and placenta; is present at low levels in skeletal muscle, kidney and brain; and is not detectable in liver. These results are consistent with the expression of cyr61 in mouse tissues.

In addition, total cellular RNA was isolated from human skin fibroblasts (HSFs) that were either quiescent, growing exponentially, stimulated by serum, or exposed to cycloheximide. HUVE cells (ATCC CRL 1730) were maintained in Ham's F12 medium supplemented with 10% fbs (Intergene), 100 p.g/ml heparin (Gibco BRL) and 30 /g/ml endothelial cell growth supplement (Collaborative Biomedical Products). Human skin fibroblasts (HSF, ATCC CRL-1475) and WI38 fibroblasts (ATCC 31 were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fbs. Quiescent HSFs were prepared by growth in DMEM supplemented wiith 1O% -fbsto 6 co6nTieice f6llowed y c-hangiing the medium to DMEM containing 0.1 fbs, for 2 days. Serum stimulation was carried out by changing the medium to 20% fbs for 1 hour. Where indicated, cycloheximide was added to 10 /g/ml simultaneously with serum for 3 'fours.

RNAs from the aforementioned cells were isolated using a guanidinium isothiocyanate protocol. Chomczynski et al., Anal. Biochem.

162.156-159 (1987). RNA samples were analyzed by electrophoretic 10 separation in fonnaldehyde-agarose gels followed by transfer to nylon filters.

Blots were hybridized with random-primed probes generated using either cyr61 or GAPDH as a template. Adams et al., Nature 355:632-634 (1992).

The results indicated that human cyr61 mRNA is not detectably present in quiescent human skin fibroblasts, is abundant in logarithmically growing and 15 senrm stimulated HSFs, and is superinduced by cycloheximide.

The analysis of RNA encoding CTGF also involved techniques that are standard in the art. In particular, investigation of RNA encoding CTGF involved the isolation of total cellular RNA and Northern analyses, performed as described in U.S. Patent No. 5,408,040, column 11, line 59, to 20 column 12, line 14, and column 13, lines 10-29, incorporated herein by reference. A 2.4 kb RNA was identified. The expression of CTGF was high in the placenta, lung, heart, kidney, skeletal muscle and pancreas. However, CTGF expression was low in the liver and brain.

Example 4 Transgenic Animals The construction of transgenic mice bearing integrated copies of recombinant cyr61 sequences was accomplished using linear DNA fragments containing a fusion gene. The cyr61 coding sequence was independently fused to the /-actin, K14, and pgk promoters, described above.

32 Expression of cyr61 was driven by these promoters in the transgenic animals.

The fusion gene was produced by appropriate restriction endonuclease digestions, using standard techniques. The fusion gene fragments were injected into single-cell zygotes of Swiss Webster mice. The injected zygotes were then implanted into pseudopregnant females. Several litters of mice were produced in this manner. Newborns exhibiting unusual phenotypes were subjected to additional analyses. For example, neonatal transgenic mice expressing cyr61 under the pgk promoter exhibited skeletal deformities, including curly tails, immobile joints, and twisted limbs, resulting in 10 locomotive difficulties. These mice typically were runted and died within seven days of birth. Transgenic mice expressing cyr61 under the 3-actin promoter showed no obvious phenotype except that the mice were smaller.

When mice bearing the transgene were back-crossed to the in-bred strain C57BL/6, the progeny mice became progressively more runted with continued S. 15 back-crossing. After three to four such back-crosses, essentially no progeny survive to reproduce. Transgenic mice expressing cyr6l under the K14 promoter exhibited a fonn of fibrotic dermatitis. The pathology involved excessive surface scratching, sometimes resulting in bleeding. Transgenic organisms having knockout mutations of cyr61 can also be created using these 20 standard techniques, Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual (Cold Spring Harbor Laboratory Press 1994), and are useful as models of disease states.

33 Example Cyr61 Expression Native Cyr61 isexpressed-in eii-bry6niic tissues and is iidiucied in a variety of wounded tissues. See below; see also, O'Brien et al. (1992).

The tissue distribution of Cyr61 was examined with rabbit anti-Cyr61 polyclonal antibodies elicited using a conventional immunological technique (Harlow et al., 1987) and affinity-purified. Using affinity-purified anti-Cyr61 polyclonal antibodies according to the invention, cyr61 expression was found in a variety of tissues, including smooth muscle, cardiomyocytes, and 10 endothelia of the cardiovascular system; brain, spinal cord, ganglia and *o .Wo' neurons, and retina of the nervous system; cartilage and bone of the skeletal system; epidermis, hair, oral epithelia, and cornea of the skin; bronchioles and blood vessels of the lung; and placental tissues. In addition to expression studies directed towards native cyr61 (mRNA and protein), studies using cyr61 15 transgenes, as described above, have contributed to our understanding of Cyr61 expression. The use of transgene fusions comprising the expression *oo" control sequences of cyr61 and the coding sequence of lacZ (encoding 3galactosidase) has provided a convenient colorimetric assay for protein S* expression.

20 The colorimetric assay involves the use of 5-Bromo-4-Chloro-3- Indolyl-/3-D-Galactopyranoside X-Gal) as a substrate for 0-galactosidase, the gene product of lacZ. Enzymatic cleavage of X-Gal by 0-galactosidase produces an intensely colored indigo dye useful in histochemical staining. In practice, embryonic and adult tissues subjected to analysis were dissected and fixed in 2% formaldehyde, 0.2% glutaraldehyde, 0.02% Nonidet P-40, and 0.01. sodium deoxycholate, in standard phosphate-buffered saline (PBS).

Fixation times varied from 15-120 minutes, depending on the size and density of organ or embryo samples being subjected to analysis. .Subsequently, samples were rinsed in PBS and stained overnight at 37°C in a PBS solution containing 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 2 34 mM MgCI,,.0,02% Nonidet P-40, 0.01% sodium deoxycholate and 1 mg/ml of X-Gal (40 mg/ml in dimethylsulfoxide [DMSO]). Samples were then rinsed in PBS, post-fixed in 4% paraformaldehyde for 1-2 hours, and stored in 70% ethanol at 40C until subjected to microscopic examination. Mice containing the cyr61-lacZ transgene were used to map the expression profile of cyr61. The results are presented in Table I for embryonic tissues at day 12.5.

Table I Transgenic Blood Skeleton Nervous Epidermis 10 Mouse Line Vessels System S2.S 3.S 4.T

NA

5.T

NA

6.T

NA

7.T

NA

8.T

NA

Transgenic lines, S- stable (established) transgenic lines; T- transient lines Expression pattern only partially reproduced.

The results indicate that Cyr61 is expressed in a variety of embryonic cell types. Additional information has been gleaned from the ectopic expression of Cyr61 resulting from another type of transgene fusion comprising a heterologous expression control sequence coupled to the coding sequence of cyr61. The control sequences, the K14 keratin promoter, the 3actin promoter, and the phosphoglycerokinase--promoter, directed the expression of Cyr61 in a pattern that differed frai its native expression.

35 Transgenic mice ectopically expressing Cyr61 were routinely smaller than wild type mice and exhibited a reduction in average life span.

Moreover, these transgenic mice-had abnormal hearts ;thickened dhaiiber walls with a corresponding reduction in internal capacity) and abnormal skeletons characterized by curved spines, joints swollen to the point of immobility, and curly tails. Therefore, ectopic expression of Cyr61 inteiferes with angiogenesis (blood vessel development and heart development) and chondrogenesis (skeletal development). In addition, transgenic mice carrying knockout mutations of cyr61 may be developed and tested as models of disease :i 10 states associated with a lack of Cyr61 activity.

A strategy for the expression of recombinant cyr61 was designed using a Baculovirus expression vector in Sf9 cells. Expression systems involving Baculovirus expression vectors and Sf9 cells are described in Current Protocols in Molecular Biology 16.9.1-16.12.6 (Ausubel et al., 15 eds., 1987). One embodiment of the present invention implemented the expression strategy by cloning the murine cyr61 cDNA into pBlueBac2, a transfer vector. The recombinant clone, along with target AcMNPV Autographa californica nuclear polyhedrosis virus, or Baculovirus)

DNA,

were delivered into Sf9 cells by liposome-mediated transfection, using the 20 MaxBac Kit (Invitrogen, Inc., San Diego, CA) according to the manufacturer's instructions. Recombinant virus was plaque-purified and amplified by 3 passages through Sf9 cells via infection.

Conditioned medium of Sf9 insect cells infected with a baculovirns construct driving the synthesis of murine Cyr61 was used as a source for purification of Cyr61 (see below). The purified recombinant Cyr61 retains certain characteristics of the endogenous protein, the heparin-binding activity of Cyr61 (described below) from 3T3 fibroblast cells and had a structure similar to the endogenous protein as revealed by independent peptide profiles produced by partial proteolysis using either 36chymotrypsin or trypsin (sequencing ,grade; Boehringer-Mannheim, Inc., Indianapolis, IN).

Human cyr61 was also expressed using the baculovirus system.

A SmnaI-HindmII fragment (corresponding to nucleotides 100-1649 of SEQ ID 5 NO: 3) of cyr61 cDNA spanning the entire human cyr61 open reading frame was subcloned into a pBlueBac3 baculovirus expression vector (Invitrogen).

Recombinant baculovirus clones were obtained, plaque purified and amplified through three passages of Sf9 infection, using conventional techniques.

Infection of Sf9 cells and human Cyr61 (hCyr61) purification was performed 10 using standard techniques, with some modifications. Sf9 cells were maintained in senum-free Sf900-II medium (Sigma). Sf9 cells were seeded, at 2-3 x 106 cells per 150 mm dish, in monolayer cultures and were infected with 5 plaque forming units (PFU) of recombinant virus per cell. The conditioned medium was collected at 8 and 96 hours post-infection, cleared 15 by centrifugation (5000 x g, 5 minutes) and adjusted to 50 mM MES Morpholino)ethanesulfonic acid], pH 6.0, 1 mM PMSF (phenylmethylsulfonyl fluoride), and 1 mM EDTA. The medium was mixed with Sepharose S beads equilibrated with loading buffer (50 mM MES, pH 6.0, 1 mM PMSF, 1 mM EDTA, 150 mM NaCI) at a ratio of 5 ml Sepharose S beads per 500 ml of 20 conditioned medium and the proteins were allowed to bind to the Sepharose S at 4°C with gentle stirring. Sepharose S beads were collected by sedimentation without stirring for 20 minutes and applied to the column. The column was washed with 6 volumes of 0.3 M NaCI in loading buffer and recombinant human Cyr61 was eluted from the column with a step gradient of NaCI (0.4-0.8 M) in loading buffer. This procedure resulted in 3-4 milligrams of purified Cyr61 protein from 500 ml of conditioned medium, and the purified Cyr61 was over 90% pure as judged by Coomassie Blue staining of SDS-gels.

In another embodiment, the complete human cyr61 cDNA is cloned into a cytomegalovirus vector such as pBK-CMV (Stratagene, LaJolla, 37 CA) using the Polymerase Chain Reaction (Hayas/hi, in PCR: The Polymerase Chain Reaction 3-13 [Mullis et al. eds., Birkhauser 1994]) and Taq Polymerase with editing-function, followed by conventional cloning techniques to insert the PCR fragment into a vector. The expression vector is then introduced into HUVE cells by liposome-mediated transfection. Recipient clones containing the vector-borne neo gene are selected using G418. Selected clones are expanded and Cyr61 expression is identified by Reverse Transcription-Polymerase Chain Reaction RT-PCR; Chelly et al., in PCR: The Polymerase Chain Reaction 97-109 [Mullis et al. eds., Birkhauser 10 1994]) or Enzyme-Linked Immunosorbent Assays ELISA; Stites et al., in Basic and Clinical Immunology 243 [Stites et al. eds., Appleton Lange 1991]) assays.

In other embodiments of the invention, Cyr61 protein is expressed in bacterial cells or other expression systems yeast) using the 15 cyr61 cDNA coding region linked to promoters that are operative in the cell type being used. Using one of these approaches, Cyr61 protein may be Sobtained in a form that can be administered directly to patients, by intravenous routes, to treat angiogenic, chondrogenic, or oncogenic disorders.

One of skill in the art would recognize that other administration routes are 20 also available, topical or local application, liposome-mediated delivery techniques, or subcutaneous, intradermal, intraperitoneal, or intramuscular injection.

38 Example 6.

Fispl2 Expression The expression of Fispl2, and a comparison of the expression characteristics of Cyr61 and Fispl2, were investigated using immunohistochemical techniques. For these immunohistochemical analyses, tissue samples (see below) were initially subjected to methyl-Carnoy's fixative methanol, 30% chloroform and 10% glacial acetic acid) for 2-4 hours.

They were then dehydrated, cleared and infiltrated in Paraplast X-tra wax at *55-56 C for minimal duration. 7 pm thick sections were collected on poly-L- 10 lysine-coated slides (Sigma), mounted and dewaxed. They were then treated with 0.03 solution of H 2 0, in methanol for 30 min. to inactivate endogenous peroxidase activity. After rehydration, sections were put in Tris-buffered saline (TBS: 10 mM Tris, pH 7.6 and 140 mM NaCI) for 15 minutes. At that point, sections were blotted to remove excess TBS with paper towels and 15 blocked with 3% normal goat serum in TBS for 10 minutes in a humid chamber. Excess buffer was then drained and primary antibodies applied.

Affinity purified anti-Cyr6l antibodies were diluted 1:50 in 3% normal goat senrm-TBS solution. Dilution for affinity-purified anti-Fispl2 antibody was 1:25. Routine control was 3 normal goat senrm-TBS, or irrelevant antibody (for example, monoclonal anti-smooth muscle cell a-actin). Specificity of staining was confirmed by incubation of anti-Cyr61 or anti-Fispl2 antibodies with an excess of the corresponding antigen on ice for at least two hours prior to applying to sections. Complete competition was observed. By contrast, cross-competition (incubation of anti-Cyr61 antibodies with Fispl2 antigen and vice versa) did not occur.

Primary antibodies were left on sections overnight at 4 C. They were then washed with TBS twice, and subjected to 30 minutes incubation with secondary antibodies at room temperature. Secondary antibodies used were goat anti-rabbit horseradish peroxidase conjugates from Boehringer- Mannheim, Inc., Indianapolis, IN (used at 1:400 dilution). Sections were 39 washed twice in TBS and chromogenic horseradish peroxidase substrate was applied for 5 minutes (1 mg/ml of diaminobenzidine in 50 mM Tris-HCI, pH 7.2 and 0.03% H 2 0 2 Sections were then counterstained in Ehrlich's haematoxylin or in Alcian blue, dehydrated and mounted in Permount.

Mouse embryos between the neural fold (E8.5) and late organogenesis (El 8.5) stages of development were sectioned and subjected to immunostaining with antigen-affinity-purified rabbit anti-Cyr61 and anti- Fispl2 antibodies. As various organs developed during embryogenesis, the presence of Cyr61 and Fispl2 was determined. Cyr61 and Fispl2 were co- 10 localized in a number of tissues and organs. A notable example is the placenta, where both proteins were readily detectable. In particular, both Cyr6 and Fispl2 were found in and around the trophoblastic giant cells, corroborating the previous detection of cyr61 mRNA in these cells by in situ hybridization (O'Brien and Lau, 1992). Both Cyr61 and Fispl2 signals in 15 immnunohistochemical staining were blocked by either the corresponding Cyr61 or Fispl2 antigen but not by each other, nor by irrelevant proteins, demonstrating specificity. In general, Cyr61 and Fispl2 proteins could be detected both intracellularly and extracellularly.

In addition to the placenta, both Cyr61 and Fispl2 were detected in the cardiovascular system, including the smooth muscle, the cardiomyocytes, and the endothelia. Both proteins were also found in the bronchioles and the blood vessels in the lung. Low levels of anti-Cyr61 and anti-Fispl2 staining could be detected transiently in the skeletal muscle. This staining is associated with connective tissue sheets, rather than myocytes; in this instance the staining pattern was clearly extracellular.

A more complex pattern of distribution was found in the epidermis and the epithelia. Both Cyr61 and Fispl2 staining could be detected in the early, single-cell layer of embryonic epidermis, as well as in later, multilayered differentiating epidermis. Fispl2 in epidermis declined to an undetectable level by the end of gestation and remained as such through adulthood, whereas Cyr61 was readily. detectable in the epidermis. In the neonate, a strong staining for Fispl2 was seen in the oral epithelia where Cyr61 staining was much weaker, while Cyr61 was found in the upper jawbone where Fispl2 was not observed. The anti-Fispl2 signal in the oral epithelia gradually increased and remained intense into adulthood. In the tongue, both Cyr61 and-Fispl2 were seen in the keratinized epithelia, although the Fispl2 staining pattern, but not that of Cyr61, excludes the filiform papillae.

Aside from the aforementioned sites of localization, Cyr61 and 10 Fispl2 were also uniquely localized in several organ systems. For example, Cyr61, but not Fispl2, was present in skeletal and nervous systems. As expected from in situ hybridization results (O'Brien and Lau, 1992), Cyr61 protein was readily detected in the sclerotomal masses of the somites, and in cartilage and bone at later stages of development. In contrast, Fispl 2 was not detectable in the skeletal system. Since correlation with chondrocytic differentiation is one of the most striking features of cyr61 expression (O'Brien and Lau, 1992), the absence of Fispl2 in the skeletal system may underscore an important difference in the biological roles of Cyr61 and S. Fispl2.. In the E14.5 embryo, Cyr61 could be detected in the ventral spinal cord, dorsal ganglia, axial muscle and sclerotome-derived cartilaginous vertebrae. Fispl2, however, was not detected in these tissues.

By contrast, Fispl2 was uniquely present in various secretory tissues. Beginning at E16.5, Fispl2 could be detected in the pancreas, kidneys, and salivary glands. In the pancreas, Fispl2 was strictly localized to the periphery of the islets of Langerhans. In the kidney, strong Fispl2 staining was seen in the collecting tubules and Henle's loops, regions where Cyr61 was not found. In the mucous-type submandibular salivary gland only collecting ducts stained for Fispl2, whereas in the mixed mucous-serous submandibular gland, both serous acini and collecting ducts stained. The signal in acini was peripheral, raising the possibility that Fispl2 is capsule- -41 associated. In simple holocrine sebaceous glands a strong acellular Fispl2 signal was detected.

In summary, Cyr61 and Fispl2 have been co-localized in the placenta, the cardiovascular system, the lung and the skin. Neither protein was detected in the digestive system or the endocrine glands. Unique localization of Cyr61 can be detected in the skeletal and central nervous system, and Fispl2 is found in secretory tissues where Cyr61 is not.

An issue closely related to protein expression concerns the metabolic fate of the expressed proteins. Members of the cysteine-rich protein 10 family have been localized. As discussed above, secreted Cyr61 is found in the ECM and on the cell surface but not in the culture medium (Yang and Lau, 1991), yet secreted Fispl2 was readily detected in the culture medium (Ryseck et al., 1991). To address the question of whether Fispl2 is also i ECM-associated, the fate of both Cyr61 and Fispl2 was followed using pulse- 15 chase experiments. Serum-stimulated, sub-confluent NIH 3T3 fibroblasts were metabolically pulse-labeled for 1 hour and chased in cold medium for various times. Samples were fractionated into cellular, ECM, and medium fractions followed by immunoprecipitation to detect Cyr61 and Fispl2. Both proteins have a similar short half-life of approximately 30 minutes in the 20 cellular fraction, which includes both newly synthesized intracellular proteins as well as secreted proteins associated with the cell surface (Yang and Lau, 1991). It should be noted that since Cyr61 is quantitatively secreted after synthesis and only a minor fraction is stably associated with the ECM, the bulk of secreted Cyr61 is cell-surface associated (Yang and Lau, 1991).

A fraction of Cyr61 was chased into the ECM where it remained stable for several hours. Newly synthesized Fispl2 was also chased into the ECM. where its half-life was only about 1 hour. A larger fraction of Fispl2 was chased to the conditioned medium, where no Cyr61 was detectable. -Fispl2 in the conditioned medium also had a short half-life of about 2 hours. Thus. whereas Cyr61 is strongly associated with the ECM, 42 Fispl2 is associated with the ECM more transiently. This result suggests that Fispl2 might be able to act at a site distant from its site of synthesis and secretion, whereas Cyr61 may act more locally.

Since many ECM proteins associate with the matrix via interaction with heparan sulfate proteoglycans, the affinity with which a protein binds heparin might be a factor in its interaction with the ECM. The results of heparin binding assays, described below, are consistent with this hypothesis.

Example 7 Protein Purification Serum-stimulated NIH 3T3 fibroblast cells were lysed to provide a source of native murine Cyr61. Yang et al. Similarly, human fibroblasts are a source of native human Cyr61.

Recombinant murine Cyr61 was purified from Sf9 cells harboring the recombinant Baculovinrs vector, described above, containing the complete cyr61 coding sequence. Although murine Cyr61 in Sf9 cell lysates formed insoluble aggregates as was the case with bacterial cell extracts, approximately 10% of the Cyr61 synthesized was secreted into the medium in a soluble form. The soluble, secreted form of Cyr61 was therefore subjected to purification.

Initially, subconfluent Sf9 cells in monolayer cultures were generated in supplemented Grace's medium (GIBCO-BRL, Inc., Grand Island, NY). Grace. Nature 195:788 (1962). The Sf9 cells were then infected with plaque-forming-units/cell of the recombinant Baculovinis vector, incubated for 16 hours, and fed with serum-free Grace's medium. These cells were expanded in serum-free Grace's Medium. The conditioned medium was collected 48 hours post-infection, although Cyr61 expression could be detected in the medium 24 hours after infection. Subsequently, the conditioned medium was cleared by centrifugation at 5000 x g for 5 minutes, chilled to 43 4°C. adjusted to 50 mM MES, pH 6.0, 2 mM EDTA (Ethylenediamine tetraacetic acid), 1 mM PMSF (Phenylmethylsulfonyl fluoride) and applied to a Sepharose-S-column (Sigma Chemical-Co.,-St. Louis, MO) at 4C (5-mi void volume per 500 ml medium). The column was washed with a buffer mM MES, pH 6.0, 2 mM EDTA, 0.5 mM PMSF) containing 150 NaCI, and bound proteins were eluted with a linear gradient of NaCI (0.2-1.0 M) in the same buffer. The pooled fractions of Cyr61 eluted at 0.6-0.7 M NaCI as a distinct broad peak. The column fractions were 90% pure, as determined by 10% SDS-PAGE followed by Coomassie Blue staining or Western analysis, 10 using techniques that are standard in the art. Yang et al.; see also, Sambrook et al., supra. For Western analysis, blots were probed with affinity-purified anti-Cyr61 antibodies as described in Yang et al., supra. After antibody probing, Western blots were stained with ECL Enhanced ChemiLuminescence) detection reagents (Amersham Corp., Arlington Heights, 15 IL). Fractions containing Cyr61 were pooled, adjusted to pH 7.5 with Tris- HCI, pH 7.5, and glycerol was added to 10% prior to storage of the aliquots at -70 0 C. Protein concentration was determined by the modified Lowry method using the BioRad protein assay kit (BioRad Laboratories, Inc., Hercules, CA). This purification procedure was repeated at least five times with similar results. The typical yield was 3-4 mg of 90% pure Cyr61 protein from 500 ml of conditioned medium.

Fispl2 was purified using a modification of the Cyr61 purification scheme (Kireeva et al., Experimental Cell Research, in press).

Serum-free conditioned media (500 ml) of Sf9 cells infected at 10 pfu per cell were collected 48 hours post-infection and loaded onto a 5-ml Sepharose S (Sigma Chemical Co., St. Louis, MO) column. After extensive washing at 0.2 M and 0.4 M NaCI, bound proteins were recovered by step elution with mM MES (pH 6.0) containing 0.5 M NaCI. Fractions containing Fispl2 of greater than 80% purity were pooled, NaCI adjusted to 0.15 M and the -44protein was concentrated 3-5 fold on a 0.5.ml Sepharose S column with elution of the protein at 0.6 M NaCI.

This purification scheme allowed the isolation of 1.5 mg of recombinant Fispl2 protein of at least 80% purity from 500 ml of seunm-free conditioned media.

CTGF was purified by affinity chromatography usinganti- PDGF cross-reactivity between CTGF and PDGF, as described in U.S. Patent No. 5,408,040, column 7, line 15, to column 9, line 63, incorporated herein by reference.

10 Example 8 :Polypeptide Characterization The murine Cyr61 protein has a M, of 41,000 and is 379 amino acids long including the N-terminal secretory signal. There is 91% amino acid sequence identity with the 381 amino acid sequence of the human protein.

Those regions of the mouse and human proteins contributing to the similarity of the two proteins would be expected to participate in the biological activities shared by the two polypeptides and disclosed herein. However, the mouse and human proteins do diverge significantly in the central portion of the proteins, where each protein is devoid of cysteines. See, O'Brien et al., Cell Growth Diff. 3:645-654 (1992). A cysteine-free region in the murine Cyr61 amino acid sequence is found between amino acid residues 164 to 226 (SEQ ID NO:2). A corresponding cysteine-free region is found in the human Cyr61 amino acid sequence between amino acid residues 163 to 229 (SEQ ID NO: More particularly, the mouse and human Cyr61 proteins are most divergent between Cyr61 amino acids 170-185 and 210-225. Other members of the ECM signalling molecule family of cysteine-rich proteins, Fispl2 (SEQ ID NO:6) and CTGF (SEQ ID NO:8), exhibit similar structures suggestive of secreted proteins having sequences dominated by cysteine residues.

45 Because murine Cyr61 contains 38 cysteines in the 355 amino acid secreted portion, the contribution of disulfide bond formation to Cyr61 tertiary structure was investigated. -Exposure of Cyr61 to -10 mM dithiothreitol (DTT) for 16 hours did not affect the ability of Cyr61 to mediate cell attachment (see below). However, Cyr61 was inactivated by heating at C for 5 minutes, by incubation in 100 mM HCI, or upon extensive digestion with chymotrypsin. These results indicate that murine Cyr61 is a heat- and acid-labile protein whose active conformation is not sensitive to reducing agents. The aforementioned structural similarities of murine and 10 human Cyr61 polypeptides suggests that human Cyr61 may also be sensitive to heat or acid, but insensitive to reducing agents. In addition, Cyr61 is neither phosphorylated nor glycosylated.

To determine if the purified recombinant murine Cyr61 described above was the same as native murine Cyr61, two additional 15 characteristics of mouse Cyr61 were determined. First, two independent protein fingerprints of recombinant and native murine Cyr61 were obtained.

0" Purified recombinant murine Cyr61 and a lysate of serum-stimulated 3T3 cells, known to contain native murine Cyr61, were subjected to limited proteolysis with either trypsin or chymotrypsin, and their digestion products were compared. Partial tryptic digests of both the recombinant protein and cell lysate resulted in two Cyr61 fragments of approximately 21 and 19 kDa.

Similarly, fingerprinting of both preparations by partial chymotrypsin digestion produced stable 23 kDa fragments from recombinant murine Cyr61 and native murine Cyr61.

Another criterion used to assess the properties of recombinant Cyr61 was its ability to bind heparin, described below. Purified recombinant murine Cyr61 bound quantitatively to heparin-sepharose at 0.15 M NaCI and was eluted at 0.8-1.0 M NaCI. This heparin binding capacity is similar to native murine Cyr61 obtained from serum-stimulated mouse fibroblasts.

Because of the similarities of the murine and human Cyr61 proteins, -46 recombinant human..Cyr6 .should exhibit properties similar to the native human Cyr61, as was the case for the murine polypeptides.

The polypeptides of the invention also extend to fragments, analogs, and derivatives of the aforementioned full-length ECM signalling molecules such as human and mouse Cyr61. The invention contemplates peptide fragments of ECM signalling molecules that retain at least one biological activity of an ECM signalling molecule, as described above.

Candidate fragments for retaining at least one biological activity of an ECM signalling molecule include fragments that have an amino acid sequence 10 corresponding to a conserved region of the known ECM signalling molecules.

For example, fragments retaining one or more of the conserved cysteine 9 residues of ECM signalling molecules would be likely candidates for ECM signalling molecule fragments that retain at least one biological activity.

Beyond the naturally occurring amino acid sequences of ECM signalling 15 molecule fragments, the polypeptides of the invention include analogs of the amino acid sequences or subsequences of native ECM signalling molecules.

ECM signalling molecule analogs are polypeptides that differ in amino acid sequence from native ECM signalling molecules but retain at S..least one biological activity of a native ECM signalling molecule, as described above. These analogs may differ in amino acid sequence from native ECM signalling molecules, by the insertion, deletion, or conservative substitution of amino acids. A conservative substitution of an amino acid, replacing an amino acid with a different amino acid of similar properties hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge, and include the following values: alanine 1.8), arginine asparagine aspartate cysteine/cystine 47 glycine glutamate glutamine histidine isoleucine leucine lysine methionine phenylalanine proline serine -threonine tryptophan tyrosine and valine It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function.

Preferably, amino acids having hydropathic indexes of 2 are substituted.

The hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a 10 polypeptide permits calculation of the greatest local average hydrophilicity of that polypeptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101, incorporated herein by reference. Hydrophilicity values for each of the common amino acids, as reported in U.S. Patent No. 4,554,101, are: alanine 15 arginine asparagine aspartate cysteine glycine glutamate glutamine histidine isoleucine leucine lysine methionine phenylalanine proline serine threonine tryptophan tyrosine and valine Substitution of amino 20 acids having similar hydrophilicity values can result in proteins retaining biological activity, for example immunogenicity, as is understood in the art.

Preferably, substitutions are performed with amino acids having hydrophilicity values within 2 of each other. Both the hyrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observatioi, amiino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly-the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties.

48 Additionally, computerized algorithms are available to assist in predicting amino acid sequence domains likely to be accessible to an aqueous solvent. These domains are known in the art to frequently be disposed towards the exterior of a protein, thereby potentially contributing to binding determinants, including-antigenic detenninants--Having-t-he-DNA -sequence in hand, the preparation of such analogs is accomplished by method:s well known in the art site-directed) mutagenesis and other techniques.

Derivatives of ECM signalling molecules are also contemplated by the invention. ECM signalling molecule derivatives are proteins or 10 peptides that differ from native ECM signalling molecules in ways other than ,primary structure amino acid sequence). By way of illustration, ECM signalling molecule derivatives may differ from native ECM signalling molecules by being glycosylated, one form of post-translational modification.

For example, polypeptides may exhibit glycosylation patterns due to 15 expression in heterologous systems. If these polypeptides retain at least one biological activity of a native ECM signalling molecule, then these polypeptides are ECM signalling molecule derivatives according to the *OO• invention. Other ECM signalling molecule derivatives include, but are not limited to, fusion proteins having a covalently modified N- or C-terminus, 20 PEGylated polypeptides, polypeptides associated with lipid moieties, alkylated polypeptides, polypeptides linked via an amino acid side-chain functional group to other polypeptides or chemicals, and additional modifications as would be understood in the art. In addition, the invention contemplates ECM signalling molecule-related polypeptides that bind to an ECM signalling molecule receptor, as described below.

The various polypeptides of the present .invention, as described above. may be provided as discrete polypeptides or be linked, by covalent bonds, to other compounds. For example, immunogenic carriers such as Keyhole Limpet Hemocyanin may be bound to a ECM signalling molecule of the invention.

-49- Example 9 Heparin Binding Assay The heparin binding assay-for native murine Cyr61, described in Yang et al., was modified for the purified recombinant murine protein.

Initially, recombinant purified Cyr61 was suspended in RIPA (Radioimmunoprecipitation assay) buffer (150 mM NaCI, 1.0% NP-40, 0.5% deoxychblate, 0.1 SDS, 50 mM Tris-HCI, pH 8.0, 1 mM phenylmethylsulfonyl fluoride).

Next, 200 Al of a 50% slurry of heparin-Sepharose CL 6B beads (Phannacia-LKB Biotechnology, Inc., Piscataway, NJ) was added to 100 dl 10 of the recombinant Cyr61 solution and incubated for 1 hour. Under these conditions, human Cyr61 was quantitatively bound to heparin-agarose.

Application of a salt concentration gradient in RIPA buffer resulted in the elution of recombinant murine Cyr61 at 0.8-1.0 M NaCI. The elution profile of the recombinant protein was similar to the elution profile for native murine Cyr61.

One might expect that Fispl2 would bind heparin with lower affinity than Cyr61, as it does not interact with the ECM as strongly as Cyr61. To examine this possibility, metabolically labeled 35 S-cysteine; 100 /PCi per 100 mm dish; ICN] cell lysates were incubated with heparin agarose beads which were subsequently washed to remove unbound proteins. Bound proteins were eluted in increasing salt concentrations. Fispl2 from cell lysates was retained on heparin agarose but was eluted by 0.2 to 0.6 M NaCI with peak elution at 0.4 M NaCI. This is in contrast to Cyr61, which was eluted at significantly higher concentrations of NaCI. This difference in heparin binding is consistent with the differing affinities of Cyr61 and Fispl2 for the ECM, suggesting that binding to heparan sulfate proteoglycans may be a primary mechaism by which both proteins associate with the ECM.

Example Receptors Human Cyr61, like murine Cyr61, was localized to the cell surface and ECM. The localization of Cyr61 to the cell surface implicated a cell surface receptor binding Cyr61. Consistent with that implication, the biological effects of Cyr61 are mediated by the cv33 integrin, or vitronectin receptor. The c,03 integrin, in association with other integrins, forms protein clusters providing focal points for cytoskeletal attachment. Cyr61 induces the formation of protein clusters, including the protein clusters containing the ,/3 3 1 0 integrin. In addition, using an in vitro assay, the biological effects of Cyr61, including Cyr61-induced cell adhesion and mitogenesis, were abolished by the addition of either one of two monoclonal antibodies- LM609 (Cheresh, Proc.

Natl. Acad. Sci. [USA] 84:6471-6475 [1987]) or anti-VnR I (Chen et al., Blood 86:2606-2615 [1995])- directed to the a,3 3 integrin. This data led to the identification of the a,03 integrin as the Cyr61 receptor.

Cyr61 induction of HUVE cell adhesion, described in Example 13 below, led to an investigation of the divalent cation-sensitive cell surface receptors expressed by HUVE cells. The cell adhesion properties of Cyr61 S..were used to identify the receptor, which is a divalent cation-sensitive cell 20 surface receptor. The ability of Cyr61 to mediate cell adhesion, coupled with the strict requirement for divalent cations in the process, indicated that Cyr61 interacts with one of the divalent cation-dependent cell adhesion molecules from the integrin, selectin, or cadherin families. Ruoslahti et al., Exp. Cell Res. 227:1-11 (1996). Using well-characterized approaches to receptor identification, a series of inhibition studies were conducted. Inhibitors, or blocking agents, of various degrees of specificity (EDTA, similar to the EGTA described above; inhibitory peptides bearing variants of the RGD (single letter amino acid code) integrin recognition motif, such as RGDS, SGDR, and RGDSPK (Ruoslalhi, et al., Science 238:491-497 [1987].

Ruioslahri, Anr. Rev. of Cell and Dev. Biol. 12.698-715 [1996]): and 51 known, .specific anti-receptor antibodies) were used to identify a Cyr61 receptor. That receptor was the av 3 integrin, also known to function as the vitronectin receptor. Confirmation of that identification was obtained by showing that antibody LM609, a specific anti-av33 integrin antibody, could block the effect of Cyr61 on cell adhesion. Integrins form a large family of heterodimeric adhesion receptors, with a broad ligand specificity range, involved in cell-cell and cell-matrix interactions. Beyond their requirement for divalent cations and their involvement in cell-matrix adhesion events [Hynes, Cell 69:11-25 (1992)], integrins also are involved in cell 10 migration [Damsky et al., Curr. Opin. Cell Biol. 4:772-781 (1992); Doerr et al., J. Biol. Chem. 271:2443-447 (1996)] and proliferation [Juliano et al., J. Cell Biol., 120.577-585 (1993); Plopper et al., Mol. Biol. Cell 6:1349-1365 (1995); and Clark er al., Science 268:233-239 (1995)], two additional processes associated with Cyr61 activity. The Uv03 integrin was found to be essential for Cyr61-mediated cell adhesion.

Characterization of CTGF binding to cells has been reported to occur through a cell surface receptor that also interacts with PDGF-BB (the BB isoforni of PDGF), as recited in U.S. Patent No. 5,408,040, column 11, ::line 10, to column 12, line 14, incorporated herein by reference. The 20 identification of the foregoing receptors permits the the design and production of molecules and which bind to the respective receptors to inhibit the activities of ECM molecules.

Example 11 Anti-ECM Signalling Molecule Antibodies Antibodies, optionally_attached to a label or to a toxin as described below, are also contemplated by the present invention. The availability of the human cyr61 cDNA sequence and the Cyr61 deduced protein sequence facilitate the implementation of methods designed to elicit 52 anti-Cyr61 antibodies -using a number of techniques that are standard in the art. Harlow et al.

In one embodiment, polyclonal antibodies directed against Cyr61 are generated. The generation of anti-Cyr61 antibodies specific for human Cyr61, for example, is optimized by designing appropriate antigens.

The human Cyr61 protein is 381 amino acids long, including the N-terinal secretory signal. As described above, human Cyr61 exhibits a 91 amino acid sequence identity with the 379 amino acid sequence of the mouse protein.

However, the mouse and human proteins diverge most significantly in the 0 central portion of the proteins, where they are devoid of cysteines (see above).

These sequence differences are exploited to elicit antibodies specific to the human Cyr61 by using as an antigen a peptide having a sequence derived from one of the divergent regions in the human protein, although antibodies directed to a conserved region are also contemplated by the invention.

15 In another embodiment of the present invention, monoclonal antibodies are elicited using intact recombinant human Cyr61 although a fragment may be used. Female BALB/c mice are inoculated intraperitoneally with a mixture of 0.25 ml recombinant human Cyr61 (5-50 micrograms), bacterially produced or produced in eukaryotic cells, and 0.25 ml complete 20 Freund's adjuvant. Fourteen days later the injections are repeated with the substitution of incomplete Freund's adjuvant for complete Freund's adjuvant.

After an additional two weeks, another injection of human Cyr61 in incomplete Freund's adjuvant is administered. About two weeks after the third injection, tail bleeds are performed and serum samples are screened for human anti-Cyr6l antibodies by immunoprecipitation with radiolabeled recombinant human Cyr61. About two months after the initial injection, mice whose sera yield the highest antibody titers are given booster injections of Cyr61 (5-50 micrograms in incomplete Freund's acdjuvant, 0. 1 ml intravenously and 0.1 ml intraperitoneally). Three days after the booster injection, the mice are sacrificed. Splenocytes are then isolated from each 53 mouse. using standard techniques, and the cells are washed.and individually fused with a myeloma cell line, the X63Ag8.653 cell line (Harlow et using polyethylene glycol, by techniques that are known in the art.

Other suitable cell lines for fusion with splenocytes are described in Harlow et al., at page 144, Table 6.2, incorporated herein by reference. Fused cells are removed from the PEG solution, diluted into a counter-selective medium Hypoxanthine-Amninopterin-Thymidine or HAT medium) to kill unfused myeloma cells, and inoculated into multi-well tissue culture dishes.

About 1-2 weeks later, samples of the tissue culture 10 supernatants are removed from wells containing growing hybridomas, and tested for the presence of anti-Cyr61 antibodies by binding to recombinant human Cyr61 bound to nitrocellulose and screening with labeled antiimmunoglobulin antibody in a standard antibody-capture assay. Cells from i positive wells are grown and single cells are cloned on feeder layers of S. 15 splenocytes. The cloned cell lines are stored frozen. Monoclonal antibodies are collected and purified using standard techniques, hydroxylapatite chromatography. In an alternative, Cyr61 peptides used as antigens, may be attached to immunogenic carriers such as keyhole limpet hemocyanin carrier .protein, to elicit monoclonal anti-Cyr61 antibodies.

20 Another embodiment involves the generation of antibody products against a fusion protein containing part, or all, of human Cyr61, including enough of the protein sequence to exhibit a useful epitope in a fusion protein. The fusion of the large subunit of anthranilate synthase TrpE) to murine Cyr61, and the fusion of glutathione S-transferase GST) to murine Cyr61, have been used to successfully raise antibodies against murine Cyr61. Yang et al. In addition, a wide variety of polypeptides, well known to those of skill in the art, may be used in the formation of Cyr61 fusion polypeptides according to the invention.

More particularly, Yang reported a TrpE-Cyr61 fusion polypeptide that was expressed from a recombinant clone constructed by 54 cloning a fragment of the murine cyr61 cDNA containing nucleotide 456 through nucleotide 951 (encoding Cyr61 amino acids 93-379) into the SacI site of the pATHI vector. Dieclnan et al., J. Biol. Chem. 260:1513-1520 (1985). The recombinant construct was transformed into a bacterial host, E. coli K12, and expression of the fusion protein was induced by addition of 25 j/g/ml indoleacrylic acid to growing cultures. Subsequently, cells were lysed and total cell lysate was fractionated by electrophoresis on a polyacrylamide gel. The fusion protein of predicted size was the only band induced by indoleacrylic acid; that band was eluted from the gel and 0 used as an antigen to immunize New Zealand White rabbits (Langshaw Farms) using techniques that are standard in the art. Harlow et al. In addition to polyclonal antibodies, the invention comprehends monoclonal antibodies directed to such fusion proteins.

In other embodiments of the invention, recombinant antibody 15 products are used. For example, chimeric antibody products, "humanized" antibody products, and CDR-grafted antibody products are within the scope of the invention. Kashmiri et al., Hybridoma 14:461-473 (1995), incorporated herein by reference. Also contemplated by the invention are antibody fragments. The antibody products include the aforementioned types of antibody products used as isolated antibodies or as antibodies attached to labels. Labels can be signal-generating enzymes, antigens, other antibodies, lectins, carbohydrates, biotin, avidin, radioisotopes, toxins, heavy metals, and other compositions known in the art; attachment techniques are also well known in the art.

Anti-Cyr61 antibodies are useful in diagnosing the risk of thrombosis, as explained more fully in Example 20 below. In addition, anti- Cyr61 antibodies are used in therapies designed to prevent or relieve undesirable clotting attributable to abnormal levels of Cyr61. Further, antibodies according to the invention can be attached to toxins such as ricin using techniques well known in the art. These antibody products according 55 to the invention are useful in delivering specifically-targeted.cytotoxins to cells expressing Cyr61, cells participating in the neovascularization of solid tumors. These antibodies are delivered by a variety of administrative routes, in pharmaceutical compositions comprising carriers or diluents, as would be understood by one of skill in the art.

Antibodies specifically recognizing Fispl2 have also "been elicited using a fusion protein. The antigen used to raise anti-Fispl2 antibodies linked. glutathione-S-transferase (GST) to the central portion of Fispl2 (GST-Fispl2), where there is no sequence similarity to Cyr61 (O'Brien 10 and Lau, 1992). A construct containing cDNA encoding amino acids 165 to 200 of Fispl2 was fused to the glutathione-S-transferase (GST) coding sequence. This was done by using polymerase chain reaction (PCR) to direct synthesis of a fragment of DNA encompassing that.fragment offispl2 flanked by a 5' BarnHI restriction site and a 3' EcoRI restriction site. The 5' primer 15 has the sequence 5'-GGGGATCTGTGACGAGCCCAAGGAC-3' (SEQ ID NO: 9) and the 3' primer has the sequence GGGAATTCGACCAGGCAGTTGGCTCG-3' (SEQID NO: 10). For Cyr61 specific antiserum, a construct fusing the central portion of Cyr61 (amino acids 163 to 229), which contains no sequence similarity to Fispl2, to GST S 20 was made in the same manner using the 5' primer 5'-GGGGATCCTGTGATGAAGACAGCATT-3' (SEQ ID NO: 11) and the 3' primer 5'-GGGAATTCAACGATGCATTTCTGGCC-3' (SEQ ID NO: 12).

These were directionally cloned into pGEX2T vector (Phannacia-LKB, Inc.) and the clones confirmed by sequence analysis. The GST-fusion protein was isolated on glutathione sepharose 4B (Phanracia-LKB, Inc.) according to manuficturer's instructions, and used to immunize New Zealand white rabbits.

For affinity purifications, antisera were first passed through a GST-protein affinity column to remove antibodies raised against GST, then through a GST- Fispl2 or GST-Cyr61 protein affinity column to isolate anti-Fispl2 or anti- Cyr61 antibodies (Harlow et al., 1988).

-56 These antibodies ;immunoprecipitated the correct size Fisp-12 protein product synthesized in vitro directed byfispl2 mRNA. The antibodies are specific for the -Fispl2 polypeptide and show no cross-reactivity with Cyr61.

Polyclonal antibodies recognizing CTGF are also known. U.S.

Patent No. 5,408,040, column 7, line 41, to column 9, line 63, incorporated by reference hereinabove, reveals an immunological cross-reactivity between PDGF and CTGF, as described above.

Example 12 10 Inhibitory peptides S" Another embodiment of the present invention involves the use

C

of inhibitory peptides in therapeutic strategies designed to inhibit the activity of the Cyr61 protein. One approach is to synthesize an inhibitory peptide

C

based on the protein sequence of Cyr61. For example, a peptide comprising an amino acid sequence that is conserved between murine Cyr61 (SEQ ID NO:2) and human Cyr61 (SEQ ID NO:4) competes with native Cyr61 for its binding sites. This competition thereby inhibits the action of native Cyr61.

For example, administration of an inhibitory peptide by well-known routes inhibits the capacity of Cyr61 to influence the cascade of events resulting in blood clots, the vascularization of tumors, or the abnormal vascularization of the eye eye disorders characterized by vascularization of the retina or the vitreous humor), etc. In particular, an inhibitory peptide prevents Cyr61 from inhibiting the action of Tissue Factor Pathway Inhibitor, or TFPI, as described below.

In an embodiment of the invention, inhibitory peptides were designed to compete with Cyr6l. These inhibitory peptides, like the antibodies of the preceding Example, exemplify modulators of Cyr61 activity, as described in the context of a variety of assays for Cyr61 activity that are disclosed herein. The peptide design was guided by sequence comparisons 57 among murine Cyr61, Fispl2, and Nov (an avian -proto-oncogene). The amino acid sequences of .several members of this family are compared in FIGURE 1. These types of sequence comparisons provide a basis for a rational design for a variety of inhibitory peptides. Some of these designed peptides, for example peptides spanning amino acids 48-68 (SEQ ID NO: 13), 115-135 (SEQ ID NO:14), 227-250 (SEQ ID NO:15), 245-270 (SEQ ID NO:16), and 310-330 (SEQ ID NO:17) of SEQ ID NO:2, have been synthesized. A comparison of the murine Cyr61 amino acid sequence and the human Cyr61 amino acid sequence reveals that similar domains from the 10 human protein may be used in the design of peptides inhibiting human Cyr61.

In addition, sequence comparisons may involve the human Cyr61 amino acid sequence; comparisons may also include the human homolog of Fispl2, Connective Tissue Growth Factor, also identified as a member of this protein family. O'Brien er al. (1992).

Inhibitory peptides may also be designed to compete with other ECM signalling molecules, Fispl2 or CTGF, for binding to their respective receptors. The design of inhibiting peptides is facilitated by the similarity in amino acid sequences among the ECM signalling molecules. In addition, inhibitory peptide design may be guided by one or more of the methods known in the art for identifying amino acid sequences likely to comprise functional domains hydrophilic amino acid sequences as external/surface protein domains; sequences compatible with a-helical formation as membrane-spanning domains). These methods have been implemented in the fonn of commercially available software, known to those of ordinary skill in the art. See the Intelligenetics Suite of Analytical Programs for Biomolecules. Intelligenetics, Inc., Mountain View, CA. Using these approaches, inhibitory peptides interfering with the biological activity of an ECM signalling molecule such as Cyr61, Fisp 12 or CTGF, may be designed. With the design of the amino acid sequence of an inhibitory peptide, production of that peptide may be realized by a variety of well-known 58 techniques including, but.not limited to, recombinant-production and chemical synthesis. Exemplary peptides that have been shown to specifically inhibit at least one biological-activity -of Cyr61 include peptides-exhibiting-the

"RGD"

motif, or motif variants such as "RGDS," "RGDSPK, "GDR," or "SGDR, (Ruoslahti, et al., Science 238:491-497 [1987], Ruoslahti, Ann. Rev. of Cell and Dev. Biol. 12.698-715 [1996]) as described in Example 10 above.

Example 13 .:Cell Adhesion Another embodiment of the invention is directed to the use of 10 Cyr61 to mediate cellular attachment to the extracellular matrix. Induction of cellular adhesion was investigated using murine Cyr61, fibronectin, and bovine serum albumin (BSA). Immunological 96-well plates (Falcon brand) were coated with 50 /l of 0.1 BSA in PBS at 4°C in the presence of 0-30 p/g/ml concentrations of murine Cyr61 or fibronectin. After two hours exposure to the coating solution, non-diluted immune or pre-immune antisera (30 dl/well), or affinity-purified anti-Cyr61 antibodies were added. For some wells, the coating mixture was adjusted to 10 mM DTT or 100 mM HCI.

SAfter .16 hours incubation, the coating solution was removed and the well surface was blocked with 1% BSA in phosphate-buffered saline (PBS) for 1 hour at room temperature. HUVE cells were plated in Ham's complete F12K medium [GIBCO-BRL, Inc.; Ham, Proc. Natl. Acad. Sci. (USA) 53:786 (1965)] at, 5 x 103-104 cells/well. Cycloheximide was added to 100 pg/ml immediately before plating and monensin was added to 1 /IM 14 hours before plating. After a 2-hour incubation at 37 0 C, the wells were washed with PBS and attached cells were fixed and stained with methylene blue. The attachment efficiency was determined by quantitative dye extraction and measurement of the extract absorbance at 650 nm. Oliver er al., J. Cell. Sci.

92:513-518 (1989).

59 HUVE cells attached poorly to-dishes treated with BSA alone, but adhered well to dishes coated with fibronectin. Murine Cyr61-coated surfaces also supported HUVE cell attachment in a dose-dependent manner, similar to fibronectin. For example, at 1 ltg/ml, Cyr61 and fibronectin yielded A 65 o values of 0.1. An A, 50 value of 0.5 corresponded to the attachment of 6 x 103 cells. At the other end of the tested concentration range, 30 .tg/ml, Cyr61 yielded an A 6 5 o of 0.8; fibronectin yielded an A 6 5 o of 0.9. Cyr61 also promoted the attachment of NIH 3T3 cells, though less effectively than fibronectin. Cyr61-mediated cell attachment can be observed 10 as early as 30 minutes after plating, as visualized by light microscopy.

The adhesion of HUVE cells on murine Cyr61-coated surfaces was specifically inhibited by anti-Cyr61 antiserum and by affinity-purified anti-Cyr61 antibodies, but not by pre-immune serum. In contrast, attachment of cells to fibronectin-coated dishes was not affected by either the anti-Cyr61 antiserum or affinity-purified anti-Cyr61 antibodies. These results show that enhancement of cell adhesion is a specific activity of the Cyr61 protein.

Furthermore, the Cyr61-mediated cell attachment was insensitive to cycloheximide or monensin treatment, indicating that Cyr61 does not actby inducing de novo synthesis of ECM components, stimulation of fibronectin, S 20 or collagen secretion. Rather, the data support the direct action of Cyr61 on cells in effecting adhesion. The Cyr61-mediated attachment of HUVE cells was completely abolished by the presence of EGTA; however, attachment was restored by the addition of CaCI, or MgSO 4 to the medium. These results indicate that the interaction between Cyr61 and its cell surface receptor requires divalent cations, consistent with the observations leading to the identification of the 0c,3 integrin as the Cyr61 receptor described in Examiiple above.

The ability of Cyr61 to promote cell adhesion, and the ability of molecules such as anti-Cyr61 antibodies to inhibit that process is exploited in an assay for modulators of cell adhesion. The assay involves a comparison 60 of cell adhesion to surfaces, plastic tissue culture wells, that are coated with Cyr61 and a suspected modulator of cell adhesion. As a control, a similar surface is coated with Cyr61 alone. Following contact with suitable cells, the cells adhering to the surfaces are measured. A relative increase in cell adhesion in the presence of the suspected modulator, relative to the level of cell adherence to a Cyr61-coated surface, identifies a promoter of: cell adhesion. A relative decrease in cell adhesion in the presence of the suspected modulator identifies an inhibitor of cell adhesion.

The identification of a Cyr61 receptor led to the development 10 of a rapid and specific ligand-receptor assay integrin binding assay) for Cyr61. Monoclonal antibody LM609 (anti-a~,.

3 has been described.

Cheresh, 1987. Monoclonal antibody JBS5 (anti-fibronectin antibody) was purchased from Chemicon. Anti-human and anti-bovine vitronectin antisera were from Gibco BRL. HRP-conjugated goat anti-rabbit antibody was from KPL. RGDSPK peptide was from Gibco BRL; RGDS and SDGR peptides were from American Peptide Company. The peptides for functional assays were dissolved in PBS at 10 mg/ml and the pH was adjusted to 7.5-8.0 with NaOH. Human plasma vitronectin was from Collaborative Biomedical Products.

20 ,,33 integrin purification from HUVE cell lysates was done as described in Pyrela et al., Meth. Enzymol., 144:475-489 (1987). Briefly, cells were lysed in 1 ml of PBS containing 1 mM CaCI, 1 mM MgCI 2 mM PMSF and 100 mM octylglucoside. The lysate was passed four times through a 0.5 ml column containing RGDSPK Sepharose (prepared from the cyanogen bromide activated Sepharose CL.4B as described in Lam, S. J.Biol. Chem., 2675649-5655 (1992). The column was washed with 10.ml of the lysis buffer and the bound protein was eluted with 2 ml of the same buffer containing 1 mM RGDS peptide at room temperature. The av,3 integrin was dialyzed against PBS containing -1 mM CaCI,, I mM MgCI 2 5 mM octylglucoside and 0.1 mM PMSF with three changes of the dialysis buffer to -61 remove the RGDS peptide. The protein was stored in aliquots at -70 0 C. The purity of the integrin was determined by SDS-PAGE under non-reducing conditions, followed by silver staining. Western -blotting :with anti-CD47 antibody showed that this ea3z integrin preparation does not contain any integrin-associated proteins.

The integrin binding assay was developed in accordance with the disclosures in Brooks et al., Cell 85:683-693 (1996), and Lam, S.C.-T.

(1992). Approximately 50 ng of the integrin in a total volume of 50 l were added per well of 96-well immunological Pro-Bind plates (Falcon) and 10 incubated overnight at 4°C. Non-specific sites were blocked with 20 mg/ml BSA in the same buffer and washed four times in that buffer. Treated plates S were incubated with 1 A/g/ml Cyr61 or 0.1 /Ag/ml vitronectin for 3 hours at room temperature. EDTA (5 mM), RGDS peptide (0.5 mM) and blocking antibodies were either preincubated with the immobilized integrin for 1 hour 15 before the addition of the protein ligand or added along with the ligand. The final dilution of the LM609 ascites fluid was 1:200. Bound proteins were detected by specific polyclonal antisera (anti-Cyr61 antisenum was diluted 1:500 and anti-vitronectin antiserum was diluted 1:1000 in PBS containing 1 mM CaCl,, I mM MgCI,, and 5 mg/mi BSA) followed by a secondary 20 antibody-horseradish peroxidase conjugate (1:20000 in the same buffer).

Plates were rinsed four times with PBS containing 1 mM CaCI, and 1 mM MgC1, after each incubation. Horseradish peroxidase (HRP) was detected with an HRP immunoassay kit (Bio-Rad Laboratories). The colorimetric reaction was developed for 15-30 minutes at room temperature, stopped by the addition of HSO 4 and the absorbance at 450 nm was measured. Those of ordinary skill in the art will understand that a variety of detection techniques could be employed in place of the enzyme-linked immunological approach exemplified. For example, other labels such as radiolabels, fluorescent compounds and the like could be bound. covalently, to an antibody or other agent recognizing the peptide of interest such as Cyr61.

-62 The results of integrin binding assays.showed that vitronectin and Cyr61 bound to the immobilized integrin. Further, both Cyr61 and vitronectin binding to c 33 were saturable. The concentration of Cyr61 at which saturation was reached was significantly higher than the concentration of vitronectin required for saturation. This difference may reflect a lower affinity of a033 for Cyr61 compared to vitronectin, which is in agreementwith the results of cell adhesion assays, which show that HUVE cells adhere to vitronectin and, more weakly, to Cyr61, in a concentration-dependent manner (see below). The specificity of the interaction was addressed by blocking the 10 ligand binding site of the integrin using any one of several techniques, including divalent cation deprivation, RGDS peptide competition, and LM609 antibody inhibition. The interaction of both proteins (Cyr61 and vitronectin) 9 with e~v3 was inhibited by EDTA, the RGDS peptide, and the LM609 antibody. These properties of the Cyr61 interaction with a,0 3 were also in o 15 agreement with the results of the cell adhesion assay and indicated that HUVE cell adhesion to Cyr6l was mediated by the direct interaction of Cyr61 with the 0,3 3 integrin.

In addition, Cyr61 induces focal adhesion, cell surface foci for cytoskeletal attachments. Focal adhesion is effected by cell surface protein 20 complexes or clusters. These protein clusters are complex, including a variety of receptors from the integrin family, and a variety of protein kinases. The induction of focal adhesion by Cyr61 is reflected in the capacity of Cyr61 to induce particular members of these cell surface protein clusters. For example, Cyr61 induces the phosphorylation of Focal Adhesion Kinase, a 125 kDa polypeptide, and Paxillin, another protein known to be involved in the focal adhesion cell surface protein complexes. Moreover, indirect immunofluorescence studies have shown that Cyr61 is bound to a receptor (see above) in focal adhesive plaques. The plaques, in turn, are characteristic of focal adhesion protein complexes. Focal Adhesion Kinase, Paxillin, and Cey, Integrin are co-localized to the focal adhesion plaques produced by focal 63 adhesion complex formation induced by Cyr61. These focal adhesion protein complexes bind Cyr61 at the cell surface; the complexes also attach internally to the cytoskeleton. Therefore, murine Cyr61, and human Cyr61 (see below), are, in part, adhesion molecules, a characteristic distinguishing Cyr61 from conventional growth factors. Those of skill in the art will also recognize that the oA integrin can be used, in conjunction with Cyr61, to screen for modulators of Cyr61 binding to its receptor. In one embodiment, the integrin is immobilized and exposed to either Cyr61 and a suspected modulator of receptor binding; or Cyr61 alone. Subsequently, bound Cyr61 is detected, 10 by anti-Cyr61 antibody that is labeled using techniques known in the art, such as radiolabelling, fluorescent labelling, or the use of enzymes catalyzing colorimetric reactions. A promoter of Cyr61 binding to its receptor would increase binding of Cyr61 (and an inhibitor would decrease Cyr61), relative to the binding by Cyr61 alone.

15 In another embodiment of the invention, the effect of murine Cyr61 on cell morphogenesis was assessed by a cell spreading assay.

Polystyrene Petri dishes were coated with 2 ml of a 10 gg/ml solution of Cyr61 or fibronectin in PBS with 0. 1% BSA and treated as described above.

A third plate was treated with BSA and served as a control. Each dish 20 received 7 x 106 cells and was incubated for 2 hours. Cell spreading was analyzed by microscopy at 100-fold magnification. The results indicate that murine Cyr61 induces HUVE cell spreading to approximately the same extent as fibronectin. The efficient attachment (see above) and spreading of cells on murine Cyr61-coated substrates indicated that Cyr61 may interact with a signal-transducing cell surface receptor, leading to a cascade of cytoskeletal rearrangements and possible formation of focal contacts. Consequently, Cyr61 and Cyr61-related polypeptides may prove useful in controlling cell adhesion, the cell adhesion events that accompany metastasizing cancer cells, organ repair and regeneration, or chondrocyte colonization of prosthetic implants, discussed below.

64 In contrast to mouse Cyr61 which mediated both HUVE cell attachment and migration, hCyr61 was found to mediate cell adhesion but not spreading of HUVE cells. Immunological plates (96-well ProBind assay plates, Falcon) were coated with 0.1-30 gg/ml hCyr61, fibronectin (Gibco BRL) or vitronectin (Gibco BRL) in phosphate-buffered saline (PBS) containing 0.1% protease-free BSA (Sigma) for 16 hrs at 4°C. The wells were blocked with 1% BSA in PBS for I hr at room temperature and washed with PBS. HUVE cells were harvested with 0.02% EDTA in PBS, washed twice with serum-free F12 medium and resuspended in serum-free F12. In 10 some experiments, fbs was added to 5-10%. Also, in experiments involving vitronectin-coated plates, endogenous vitronectin was removed from fbs by imnmunoaffinity chromatography using bovine polyclonal anti-vitronectin antibodies (Gibco). Norris et al., J. Cell Sci. 95:255-262 (1990). Cells were plated at 104 cells/well. After 2 hours, cells were fixed with 4% 15 paraformaldehyde, stained with methylene blue and quantified as described.

Oliver et al., J. Cell Sci. 92:513-518 (1989).

Under serum-free conditions, hCyr61 mediated cell attachment but not spreading of HUVE cells. Attachment of HUVE cells to hCyr6l- Scoated plates was enhanced by inclusion of serum in the culture medium. In 20 the presence of serum. HUVE cells attached and spread on hCyr61 in a manner similar to that seen on fibronectin. Human Cyr61 supported HUVE cell adhesion in a dose-dependent manner both under high-serum and low-serum conditions. However, in the presence of 10% fbs, the maximal proportion of the cells attaching at a lower concentration of hCyr61.

and the proportion of the cells attached, was higher. Human Cyr61 was also found to cooperate with vitronectin in promoting HUVE cell adhesion and spreading. Two major cell-adhesive proteins found in mammalian sera are fibronectin and vitronectin, also known as "serum spreading factor." For review, see Felding-Habernann et aI., Curr. Opin. Cell Biol. 5:864-868 (1993). Cell attachment, spreading and growth on tissue-culture plastic 65 depended upon vitronectin, rather-than fibronectin, in-serum for the following reasons: considerable depletion of fibronectin in the batches of fbs due to "clotting" at 4°C; and inability of fibronectin to efficiently coat the plastic in the presence of an excess amount of other serum proteins. In contrast, vitronectin coated the plastic surfaces efficiently under the same conditions.

The ability of HUVE cells to adhere to hCyr61-coated plats in the presence of mock-immunodepleted fbs and serum immunodepleted with anti-bovine vitronectin antibodies were compared. HUVE cells adhered to hCyr61-coated surfaces significantly better in the presence of soluble 0 vitronectin or mock-immunodepleted fbs than they did in the presence of serum-free medium or medium supplemented with vitronectin-immunodepleted fbs. The addition of vitronectin (30 /g/ml) to vitronectin-immunodepleted *serum restored the ability of HUVE cells to adhere and spread on hCyr61coated plates to the same level observed when whole serum was used in the 15 cell attachment assay. Furthermore, soluble vitronectin alone, at a concentration equal to its level in 10% serum (30 restored the level of cell adhesion and spreading to the level found in the presence of serum. Thus, vitronectin is a necessary and sufficient serum component .contributing to HUVE cell adhesion and spreading on hCyr61-coated plastic surfaces. Control studies showed that the effect of vitronectin was not due to its preferential retention on the plastic dish surfaces in the presence of hCYR61.

Additionally, HUVE cell attachment and spreading in the presence of an increasing quantity of vitronectin was examined. The solutions for coating the dishes contained increasing amounts of vitronectin (0-10 Ag/ml) with a fixed amount of hCyr61 (10 The results indicated that more cells adhered to plates coated with the two proteins than would have been expected by adding the individual adhesive capacities of vitronectin and hCyr61. This non-additive increase of adhesion in the presence of vitronectin and hCyr61 was not due to higher amounts of vitronectin absorbed on the -66plastic. ELISA assay with anti-human vitronectihi antibodies showed that the amount of vitronectin adsorbed to plastic dishes exposed to the vitronectin/hCyr61 mixture did not exceed that of vitronectin alone by more than 20%. This difference is insufficient to explain the observed difference in cell adhesion (3-5 fold in different experiments). In addition, a higher proportion of HUVE cells also adhered to the mixture of proteins when' the coating solution contained diluted vitronectin (2.5 /g/ml) than were found to adhere to dishes coated with higher concentrations of pure vitronectin g/ml) or pure hCyr61 (10 jg/ml). Thus, vitronectin and hCyr61 functionally 0 cooperate and exert a synergistic effect on HUVE cell adhesion.

e* The capacity of Fispl2 to affect cell adhesion was also investigated. Fispl2 cell attachment assays were performed essentially as So described (Oliver et al., 1989). 96-well immunological plates were coated for 16 hours at 4 0 C with 20 j/g/ml Cyr61, Fispl2 or fibronectin (Gibco BRL) in 15 PBS containing 0.1 mg/ml BSA and blocked with 10 ing/ml BSA for 1 hour at room temperature. HUVE cells were plated at 104 cells/well in F12K media with 10% FBS (Hyclone Laboratories, Inc., Logan, Utah); NIH 3T3 fibroblasts were plated at 3 x 10' cells/well and MvlLu cells were plated at x 10' cells/well in minimal essential medium (MEM) with 10% FBS. After 1 hour incubation cells were fixed, stained with methylene blue and quantified as described (Oliver et al., 1989). Cell spreading was examined on cells plated on 100 mm polystyrene petri dishes coated with 2.5 ml of a 20 /g/ml solution of Cyr61, Fispl2 or fibronectin. 107 cells were plated on each dish and cell spreading was analyzed 90 min. after plating by microscopy at 100X magnification.

The results indicated that Fispl2, as well as Cyr61, when coated on plastic dishes, promoted the attachment of three different cell types: HUVE cells, NIH 3T3 fibroblasts, and mink lung epithelial (MvlLu) cells.

These cells attached poorly to uncoated plastic dishes or plastic dishes coated with bovine serum albumin, but attached significantly better to dishes coated 67with either fibronectin, Cyr61, or Fispl2. The ability of either Cyr61 or Fispl2 to mediate cell attachment is comparable to that of fibronectin for all three cell types. While the ability of Cyr61 to mediate cell attachment was previously demonstrated for fibroblasts and endothelial cells (Kireeva et al., 1996), these studies show cell attachment activity for both Fispl2 and Cyr61 in epithelial cells in addition to endothelial cells and fibroblasts.

Like cell attachment to fibronectin and Cyr61 (Kireeva et al., 1996), Fispl2-mediated cell attachment was inhibited when EDTA was added to the culture medium. This inhibition was completely abolished by the 10 addition of excess MgCl 2 indicating a requirement for divalent cations in Fispl2-mediated cell attachment. In addition to cell attachment, Fispl2 also promotes cell spreading. Similar cell spreading was found when NIH 3T3 cells were plated on dishes coated with either fibronectin, Cyr61, or Fispl2, but not BSA. Endothelial and epithelial cells also spread when plated on

S

fibronectin, Cyr61, or Fispl2.

Example 14 Migration of Fibroblasts Cyr61 also affects chondrocytes, fibroblasts involved in skeletal development. In particular, Cyr61 influences the development, and perhaps maintenance, of cartilage, in contrast to the variety of growth-related proteins that exclusively influence development and maintenance of the bony skeleton. The chemotactic response of NIH 3T3 cells to murine Cyr61 was examined using a modified Boyden chamber (Neuroprobe Inc., catalog no.

AP48). Grotendorst, Meth. Enzymol. 147:144-152 (1987). Purified Cyr61 protein was serially diluted in DMEM containing bovine serum albumin (BSA; 0.2 mg/ml) and added to the lower well of the chamber. The lower well was then covered with a collagen-coated polycarbonate filter (8 /m pore diameter; Nucleopore Corp., Pleasanton, CA). Cells (6 x 104) were then loaded into the upper well. After 5 hours incubation (10% CO,, 37 0 the filter was 68 removed and -the cells -were fixed and stained using Wright-Giemsa stain (Harleco formulation; EM Diagnostic Systems, Gibbstown, NJ). Cells from the upper surface of the filter were then removed by wiping with a tissue swab. The chemotactic response was determined by counting the total number of migrating cells detected in ten randomly selected high-power microscopic fields (400-fold magnification) on the lower surface of the filter. Duplicate trials were performed for each experiment and the experiment was repeated three times to ensure reproducibility of the data.

NIH 3T3 cells responded to Cyr61 as a chemotactic factor in a dose-dependent manner in the Boyden chamber assay. Without Cyr61, approximately 4.8 cells had migrated per high-power field. In the presence of 0.5 /g/ml murine Cyr61, about 5.2 cells were found in each field. As the concentration of Cyr61 was raised to 1, 5 and 10 Ag/ml, the average number of migrating cells detected per field rose to 7.5. 18.5, and 18.7. Thus, S 15 murine Cyr61 acts as a chemoattractant for fibroblasts. The optimal concentration for the chemotactic activity of Cyr61 is 1-5 /g/ml in this assay; this concentration range is consistent with the reported ranges at which other ECM molecules provide effective chemotactic stimulation. For example, Thrombospondin, at 5-50 tg/ml, has a chemotactic effect on endothelial cells 20 (Tarabolerti et al., J. Cell Biol. 111:765-772 (1990); fibronectin also functions as a chemotactic agent at 1-30 /ig/ml (Carsons er al., Role of Fibronectin in Rheumatic Diseases, in Fibronectin [Mosher, ed., Academic Press 1989]; Carsons et al., Arthritis. Rheum. 28:601-612 [1985]) as determined using similar Boyden chamber assays. The human Cyr61 polypeptide may be used to chemoattract fibroblasts in a manner analogous to murine Cyr61. Human CTGF has also been reported to induce the migration of non-human mammalian cells such as NIH 3T3 cells (mouse fibroblasts) and BASM cells (bovine aortic smooth muscle cells), as described in U.S. Patent No.

5,408,040, column 7, line 65 to column I line 7, incorporated herein by reference.

-69- In an alternative embodiment, an assay for.modulators of cell migration, such as the migration of chondrocytes, involves a combination of a suspected modulator of cell migration and Cyr61 being added to the lower well of a Boyden chamber. As a control, Cyr61 is separately added to the lower well of another Boyden chamber. Relative cell migrations are then measured. An increase in cell migration in the presence of the suspected modulator relative to cell migration in response to Cyr61 alone identifies a promoter of chondrocyte cell migration, while a relative decrease in cell migration in the presence of the suspected modulator identifies an inhibitor.

Example Migration of Endothelial Cells- In Vitro Assays The end product of in virro angiogenesis is a well-defined network of capillary-like tubes. When cultured on gel matrices, e.g., collagen, fibrin, or Matrigel gels, endothelial cells must first invade the matrix before forming mature vessels. (Matrigel is a complex mixture of basement membrane proteins including laminin, collagen type IV, nidogen/entactin, and proteoglycan heparin sulfate, with additional growth factors. Kleinman et al., S Biochem. 25:312-318 (1986). The invasive structures are cords which eventually anastomose to form the vessel-like structures. The angiogenic effect of human Cyr61 on confluent monolayers of human umbilical vein endothelial cells is assessed by seeding the cells onto three-dimensional collagen or fibrin gels, in the presence or absence of Cyr61. HUVE cells do not spontaneously invade such gels but do so when induced by agents such as tumor promoters.

Collagen gels were prepared by first solubilizing type I collagen (Collaborative Research, Inc., Bedford, MA) in a sterile 1:1000 dilution of glacial acetic acid (300 ml per gram of collagen). The resulting solution was filtered through sterile triple gauze and centrifuged at 16,000 x g for I hour at 4°C. The supernatani was dialyzed against 0. X Eagle's Minimal 70 Essential Medium .(MEM; GIBCO-BRL, Inc,) and stored at 4°C. Gels of reconstituted collagen fibers were prepared by rapidly raising the pH and ionic strength of the collagen solution. The pH and ionic strength adjustments were accomplished by quickly mixing 7 volumes of cold collagen solution with one volume of 10X MEM and 2 volumes of sodium bicarbonate (11.76 mg/ml) in a sterile flask. The solution was kept on ice to prevent immediate gelation.

The cold mixture was dispensed into 18 mm tissue culture wells and allowed to gel for 10 minutes at 37 0

C.

Fibrin gels were prepared by dissolving fibrinogen (Sigma 10 Chemical Co., St. Louis, MO) immediately before use in calcium-free MEM to obtain a final concentration of 2.5 mg of protein/ml. Clotting was initiated by rapidly mixing 1.35 ml of fibrinogen solution with 15 /l of 10X MEM containing 25 U/ml thrombin (Sigma Chemical Co.) in a plastic tube. The mixture was transferred immediately into 18 mm tissue culture wells and allowed to gel for about 2 minutes at 37 0

C.

In some wells, Cyr61 was mixed into the gel matrix before gelation (final concentration 10 jg/ml), while in other wells, Cyr61 was not in the gel matrix but was added as part of the nutrient medium (similar range of concentrations as in the matrix) after the cells reached confluency. HUVE 20 cells were seeded onto the gel matrix surface at 5 x 10' cells per well in Ham's F12K medium [GIBCO-BRL, Inc.] containing 10% fetal bovine serum, 100 ug/ml heparin, and 30 j/g/ml endothelial cell growth factor. When the cells reached confluency, the medium was removed, the cells were rinsed with PBS, and fresh medium without endothelial cell growth factor was supplied.

Some cultures received purified recombinant Cyr61, while others received Cyr61 and polyclonal anti-Cyr61 antibodies. Thus, the variety of cultures at confluency included: a) cultures with no Cyr61; h) cultures with Cyr61 within the matrix; c) cultures with Cyr61 supplementing the medium; and d) cultures with Cyr61 supplementing the medium along with polyclonal anti-Cyr61 antibodies.

-71 Invasion of,the gel matrix was quantified about 4-7 days after treatment of the confluent cultures. Randomly selected fields measuring mm x 1.4 mm were photographed in each well under phase-contrast microscopy with a Zeiss Axiovert inverted photomicroscope. Photographs were taken at a single level beneath the surface monolayer. Invasion was quantified by measuring the total length of all cell cords that penetrated beneath the surface monolayer. Results were expressed as the mean length in microns per field for at least 3 randomly selected fields from each of at least 3 separate experiments.

10 In order to examine the network of cords within the matrix for capillary-like tube formation, cultures were fixed in situ overnight with glutaraldehyde and 1% tannic acid in 100 mM sodium cacodylate buffer, pH 7.4. Cultures were then washed extensively in 100 mM sodium cacodylate buffer, pH 7.4. The gels were cut into 2 mm x 2 mm fragments, post-fixed 15 in I osmium tetroxide in veronal acetate buffer (to minimize tissue swelling; see Hayar, in Principles and Techniques of Electron Microscopy: Biological Applications 1:38 [Litton Educational Publishing, Inc. 1970]) for 45 minutes, stained en bloc with 0.5 uranyl acetate in veronal buffer for 45 minutes, dehydrated by exposure to a graded ethanol series, and embedded in Epon in flat molds. Semi-thin sections were cut perpendicular to the culture plane with an ultramicrotome, stained with 1% toluidine blue, and photographed under transmitted light using an Axiophot photomicroscope (Zeiss).

In an alternative embodiment, a suspected modulator of angiogenesis is combined with Cyr61 and the combination is added before, or after, formation of a gel. In this embodiment, a control is established by using Cyr61 alone. The migration of cells in response to the suspected modulator and Cyr61 is then compared to the migration of cells in response to Cyr61 alone. A promoter or positive effector will increase cell migration while an inhibitor or negative effector will decrease cell migration.

72 In an alternative in vitro assay for angiogenic activity, an assay for endothelial cell migration was developed. This chemotaxis assay has been shown to detect the effects of Cyr61 concentrations on the order of nanograms per milliliter. Primary Human Microvascular Endothelial Cells (HMVEC P051; Clonetics, San Diego, CA) were maintained in DME with 10% donor calf serum (Flow Laboratories, McLean, VA) and 100 jg/ml endotheliakcell mitogen (Biomedical Technologies Inc., Stoughton, MA). The cells were used between passages 10 and 15. To measure migration, cells were starved for 24 hours in DME containing 0.1 BSA, harvested, resuspended in DME 10 with 0.

1 BSA, and plated at 1.75 x 10' cells/well on the lower surface of a gelatinized 0.5 /mi filter (Nucleopore Corporation, Pleasanton, CA) in an inverted modified Boyden chamber. After 1-2 hours at 37°C, during which time the cells were allowed to adhere to the filter, the chamber was reverted to its normal position. To the top well of separate chambers, basic Fibroblast S* 15 Growth Factor (a positive control), Cyr61, or a negative control solution (conditioned medium known to lack chemoattractants or DME plus BSA, see below) was added at concentrations ranging from 10 ng/ml to 10 zg/ml.

Chambers were then incubated for 3-4 hours at 37 0 C to allow migration.

Chambers were disassembled, membranes-fixed and stained, and the number of cells that had migrated to the top of the filter in 3 high-powered fields was determined. Tolsma et al., J. Cell. Biol. 122:497-511 (1993) (incorporated by reference), and references cited therein. DME with 0.1 BSA was used as a negative control and either bFGF (10 ng/ml) or conditioned media from angiogenic hamster cell lines (20 Ag/ml total protein) were used as positive controls. Rastinejad et al., Cell 56:345-355 (1989). Each sample was tested in quadruplicate (test compound such as Cyr61, positive control, conditioned medium as a negative control, and DME plus BSA as a negative control) in a single experiment; experiments were repeated at least twice.

To allow comparison of experiments performed on different days, migration data is reported as the percent of maximum migration towards 73 the positive control, calculated after subtraction of background migration observed in the presence of DME plus BSA. Test compounds that depressed the random movement of endothelial cells showed a negative value for the percent migration. Very high concentrations of thrombospondin (TSP) caused endothelial cells to detach from the membrane. Detachment was detected by counting cells on the lower face of the membrane. When cell loss exceeded the number of migrated cells was corrected for this loss. The results indicate that 0.01-10 ,g/ml bFGF induced the migration of a constant 92 cells per three high-powered microscope fields Migration in the presence of Cyr61 10 revealed a greater dependence on concentration. At 10 ng/ml, Cyr61 induced S.e. an average of 64 cells to migrate per three high-powered fields examined. At 100 ng/ml Cyr61, approximately 72 cells were found in three fields; at 1 /Ag/ml Cyr61, a peak of 87 cells had migrated; at approximately 7 /g/ml Cyr61, about 61 cells were observed; and at 10 pg/ml Cyr61, approximately 57 cells were found to have migrated. The negative control revealed a constant basal level of endothelial cell migration of 53 cells per three highpowered microscope fields. In addition to these results, there is a perfect correlation of the results from this in vitro assay and the results from the in vivo cornea assay, described below.

To monitor toxicity, endothelial cells were treated with each of the tested compounds at a range of concentrations, under conditions identical to those used in the migration assay. Cells were then stained with Trypan blue and cells excluding Trypan blue were counted. The results showed that cells remained viable and that the inhibition of migration could not be attributed to toxicity. Where relevant, endothelial cells were pretreated for 36-48 hours with peptides at 20 /M in DME with 0.1 BSA before use in the migration assays. Toxicity was also tested over these time frames and found to be negligible.

The ability of Cyr61 to induce matrix invasion and tube formation by HUVE cells, as well as the ability of Cyr61 to induce human 74 microvascular endothelial cells :to -migrate, demonstrates the angiogenic properties of this protein. It is anticipated that other members of the ECM signalling molecule family of cysteine-rich proteins, such as Fispl2 and CTGF, have similar properties that may be used in methods of the invention for screening for, and modulating, angiogenic conditions. In particular, one of ordinary skill in the art understands that an in vitro assay for angiogenic inhibitors involves the assay described above, including an effective amount of Cyr61, with and without the candidate inhibitor.

Example 16 -1 I0 Migration of Endothelial Cells- An In Vitro Assay For Angiogenesis Inhibitors The inclusion of an effective amount of an ECM signalling molecule, such as Cyr61, in the in vitro migration chemotaxis) assay described in the preceding Example, provides an assay designed to detect inhibitors of ECM signalling molecules and angiogenesis. Because of the crucial role of neovascularization in such processes as solid tumor growth and metastasis, the development of assays to detect compounds that might antagonize these processes would be useful.

~The above-described in vitro migration assay was adapted to include an ECM signalling molecule, Cyr61. Cyr61 was included at 1 t.g/ml, which was found to be the optimal dosage in titration studies. As in the preceding Example, human microvascular endothelial cells (Clonetics) were used. In one series of assays, several carbohydrates and carbohydrate derivatives were analyzed. These compounds included 10 mM mannose, mM mannose-6-phosphate, and 10 mM galactose. Results of these assays showed that Cyr61 plus mannose yielded approximately 73 cells per set of three high-powered microscope fields (see above). Cyr61 plus galactose_ induced the migration of approximately 74 cells per set of three high-powered fields. However, Cyr61 plus mannose-6-phosphate yielded approximately 2 migrating cells for each set of three high-powered fields examined. Control 75 experiments-demonstrate that the inhibition of Cyr61 activity by mannose-6phosphate is specific.

The angiogenic activity of basic FGF (10 ng/ml) was also tested, as described above, with and without mannose-6-phosphate. In the presence of 10 mM mannose-6-phosphate, bFGF induced 51 cells per set of three high-powered fields to migrate; in its absence, bFGF induced the migration of approximately 52 cells. However, when either Cyr61 or Insulin Growth Factor II (IGF-II) were tested, mannose-6-phosphate reduced the number of migrating cells from approximately 48 or 47 cells, respectively, to 10 approximately 12 or 11 cells, respectively. The effect of mannose-6phosphate on IGF II activity was anticipated because mannose-6-phosphate is known to compete with IGF II for their common receptor (the IGF II receptor). Thus, mannose-6-phosphate specifically inhibits the chemotactic activity of Cyr61 on human endothelial cells. Moreover, because there is an 15 essentially perfect correlation between the in vitro migration assay and the in vivo angiogenesis assay, described below, mannose-6-phosphate has been identified as an inhibitor of angiogenesis based on the results of the assay disclosed herein. Accordingly, the invention contemplates a method of inhibiting angiogenesis comprising the step of administering an inhibitor the angiogenic activity of Cyr 61 such as mannose-6-phosphate. Assays such as that described above may also be used to screen for other inhibitors of angiogenesis which may be useful in the treatment of diseases associated with angiogenesis such as cancer, and diseases of the eye which are accompanied by neovascularization.

In an embodiment of the invention, a method of screening for modulators of angiogenesis involves a comparative assay. One set of conditions involves exposure of cells to a combination of Cyr61 and a suspected modulator of cell migration. As a control, a parallel assay is performed that exposes cells to Cyr61 alone. A promoter of cell migration elevates the rate of in vitro cell migration relative to the rate of migration in 76 the presence of Cyr61 alone; the converse is -true for an inhibitor of the chemoattracting ability of Cyr61.

Example 17 Migration of Endothelial Cells- An In Vivo Assay An in vivo assay for endothelial cell migration has also been developed. In general, the assay protocol is consistent with the disclosure of Tolsma et al., 1993. To assess angiogenesis associated with the formation of granulation tissue the newly fonned, proliferative, fibroblastic dermal tissue around wounds during healing), sponge implants were used as 10 previously described (Fajardo, et al., Lab. Invest. 58:718-724 [1988]).

Polyvinyl-alcohol foam discs (10-mm diam x 1-mm thick) were prepared by first removing a 2-mm diameter central core of sponge. PBS or an RGDS .peptide (other possible test compounds include fragments of Cyr61, RGDS peptide, small molecules such as mannose-6-phosphate) at 100 J/M were added to the sponge core which was then coated with 5j4l of sterile Hydron (Interferon Sciences, New Brunswick, NJ). After solidifying, the coated core was returned to the center of the sponge which was then covered on both sides with 5 gm filters and secured in place with glue (Millipore Corp., Bedford, MA). One control and one test disc were then implanted subcutaneously in the lower abdomen of anesthetized Balb/c female mice where granulation tissue could invade the free perimeter of the disc. Wounds were closed with autoclips and animals left undisturbed until sacrificed.

Quantitative estimates of thymidine incorporation in situ into endothelial cells in the discs were obtained as previously described (Polverini, et al., J. Immunol. 118:529-532 [1977]). Sponge implants were evaluated at days 5, 7, 10, and 14 after implantation. Thirty minutes before sacrifice, mice were injected with a solution containing 3 H]-thymidine in saline (specific activity 6.7 Ci/mM; New England Nuclear/Du Pont, Wilmington, DE) to a level of I MCi per gram of body weight. Sponges were removed and facially 77 embedded to -provide a uniform -section of the entire- circumference. Tissues were fixed in 10% neutral buffered formalin, dehydrated through a graded series of alcohols, and embedded in glycol methacrylate (Polysciences, Miles, IL). Autoradiograms were prepared by dipping sections mounted on acidcleaned glass slides into NTB type 2 emulsion (Eastman Kodak). After exposure for 4 weeks at 4 0 C, autoradiographs were developed in half strength D-19 developer, fixed in Kodak Rapid Fixer, and stained with hematoxylin and eosin. Quantitation of endothelial cell labeling was performed by counting all endothelial cells that lined capillaries and venules extending from 10 the periphery to the center of the sponge by rectilinear scanning under oil immersion (xl,000). A total of 500-700 endothelial cells were counted in each of two sponges containing either PBS, TSP, or peptide fragments thrombospondin fragments). Cells were considered labeled if five or more grains were detected over the nucleus. The percentage of labeled cells was 15 calculated and a chi-square analysis of data derived from control and experimental sponges was performed.

The results of the foregoing assay established that thrombospondin fragments could inhibit the process of angiogenesis. More generally, one of ordinary skill in the art would appreciate that the scope of 20 the present invention extends to such in vivo assays for suspected modulators of ECM signalling molecule activities, such as the chemotactic ability of Cyr61 to induce cell migration. As with other assays of the invention, a comparative assay involves exposure of cells, in vivo, to a sponge laden with Cyr61 in the presence or absence of a suspected modulator of Cyr61 activity.

Following implantation, incubation, and removal, the relative rates of cell migration are determined. A promoter of Cyr61 activity will increase the rate of cell migration relative to cell migration induced by Cyr61 alone; an inhibitor will decrease the rate of cell migration relative to the level ascribable to Cyr61 alone.

78 Example 18 Mitogen Potentiation In another aspect of the invention; murine Cyr61 enhanced the mitogenic effect of growth factors on fibroblasts and endothelial cells. When NIH 3T3 fibroblasts or HUVE cells were treated with a non-saturating dose of either basic Fibroblast Growth Factor (bFGF) or Platelet-Derived Growth Factor (PDGF-BB), the addition of murine Cyr61 significantly increased the incorporation of radiolabeled thymidine compared to cells treated with the growth factors alone. The thymidine incorporation assay is a standard 10 technique for detennining whether cells are actively growing by assessing the *'extent to which the cells have entered the S phase and are synthesizing DNA.

.0 The Cyr61 enhancement of bFGF- or PDGF-BB-induced thymidine incorporation was dose dependent, requiring a minimum concentration of 1.0 Ag/ml of recombinant protein for either cell type. The enhancement of 15 DNA synthesis by Cyr61 was inhibited by the addition of specific anti-Cyr61 antiserum.

More specifically, NIH 3T3 fibroblast cells were plated on 24-well plates at 3 x .104 cells/well and grown in DMEM with 10% fetal Sbovine serum (Intergen Co., Purchase, NY) for 3-4 days and incubated with 20 medium containing 0.2% FBS for the following 48 hours. The following compounds, at the parenthetically noted final concentrations, were then added to the plated cells in fresh DMEM containing 0.2 fbs and 3 H]-thymidine (1 A/Ci/ml final concentration; ICN Biomedicals, Inc., Costa Mesa, CA): bFGF ng/ml), PDGF-BB (30 ng/ml), and murine Cyr61 (0.5-5 lg/ml). These compounds were added to individual plates according to the following pattern: 1) no supplementation; 2) murine Cyr61; 3) bFGF; 4) murine Cyr61 and bFGF; 5) PDGF-BB; and 6) murine Cyr61 and PDGF. After 18-20 hours of incubation, cells were washed with PBS and fixed with 10% trichloroacetic acid. DNA was dissolved in 0.1 N NaOH and thymidine incorporation was determined. The results indicated that murine Cyr61, in the absence of a growth factor, did not stimulate DNA synthesis as measured by tritiated 79 thymidine incorporation. Without any supplements, 3T3 cells incorporated approximately 1.8 x 104 cpin of 3 H]-thymidine, in the presence or absence of Cyr61. Cells exposed to bFGF alone incorporated about 1.2 x 105 cpm; cells contacting bFGF and murine Cyr61 incorporated 2 x 105 cpm. Cells receiving PDGF-BB incorporated about 1.2 x 10.cpm; and cells exposed to PDGF-BB and murine Cyr61 incorporated approximately 2.4 x 105 cpm. Therefore, murine Cyr61 'did not function as a mitogen itself, but did potentiate the mitogenic activity of bFGF and PDGF-BB, two known growth factors.

The ability of murine Cyr61 to potentiate the mitogenic effect 10 of different levels of bFGF also revealed a threshold requirement for the *o growth factor. Human umbilical vein endothelial cells were plated essentially as described above for 3T3 cells and exposed to a constant amount of murine Cyr61; controls received no Cyr61. Different plates were then exposed to different levels of bFGF, comprising a series of bFGF concentrations ranging 15 from 0-10 ng/ml. Following culture growth in the presence of 3 H]-thymidine for 72 hours, cells exposed to 0-0. 1 ng/ml of bFGF exhibited a baseline level of thymidine incorporation (approximately 4 x 102 cpm), in the presence or absence of Cyr61. At I ng/ml bFGF, however, HUVE cells increased their thymidine incorporation in the presence of bFGF to 6 x 102 cpm; in the 20 presence of I ng/ml bFGF and murine Cyr61, HUVE cells incorporated 1.3 x 10' cpm. At 10 ng/ml bFGF, cells exposed to bFGF incorporated about 1.8 x 103 cpm thymidine; cells receiving 10 ng/ml bFGF and Cyr61 incorporated approximately 6.1 x 103 cpm.

The capacity of murine Cyr61 to potentiate the mitogenic activity of bFGF was verified by a thymidine incorporation assay involving HUVE cells and various combinations of bFGF, Cyr61, and anti-Cyr61 antibodies. Cells were plated and grown as described above. The following combinations of supplements (final plate concentrations noted parenthetically) were then pre-incubated for 1 hour before addition to individual plates: 1) preimmune antisenum 2) bFGF (15 ng/ml) and pre-immune antiserum 80 (3 3) pre-immune antiserum and Cyr61 pg/mi); 4) pre-immune antiserum Cyr61 (4 j.g/ml), and bFGF (15 ng/ml); 5) anti-Cyr61 antiserum 6) anti-Cyr61 antiserum and bFGF (15 ng/mi); 7) anti-Cyr6 antiserum and Cyr61 (4 jg/ml); and 8) anti-Cyr61 antiserum Cyr61 (4 j.g/ml), and bFGF (15 ng/ml).

Following incubation in the presence of 3 H]-thymidin e as described above, cells exposed to pre-immune antiserum incorporated about 2 x 10? cpm thymidine; cells contacting pre-immune antiserum and bFGF 0 0 incorporated 1.3 x 10' cpm; cells receiving pre-immune antiserum and Cyr61 10 incorporated 1 x 102 cpm; cells receiving pre-immune antisenm, Cyr61, and *0 @o bFGF incorporated 3.6 x 10' cpm; cells exposed to anti-Cyr6l antiserum incorporated 2 x 102 cpm; cells receiving anti-Cyr61 antiserum and bFGF incorporated about 1.3 x 10' cpin; cells contacting anti-Cyr61 antiserum and Cyr61 incorporated about 1 x. 102; and cells receiving anti-Cyr61 antisenrum, 15 Cyr61, and bFGF incorporated 1 x 103 cpm. These results indicate that preimmune antiserum had no effect on Cyr61-induced potentiation of bFGF mitogenic activity. Anti-Cyr6l antiserum, however, completely abolished the s potentiation of bFGF by Cyr6 1. Moreover, the effect of anti-Cyr61 antiserum was specific to Cyr61-induced mitogenic potentiation because anti-Cyr61 20 antiserum had no effect on the mitogenic activity of bFGF per se. Therefore, Cyr61 can be used as a reagent to screen for useful mitogens.

DNA synthesis for HUVE cells and NIH 3T3 fibroblasts was measured by thymidine incorporation as described previously (Kireeva et al., Mol. Cell. Biol. 16: 1326-1334 [1996]) with minor modifications. HUVE cells were grown in 24-well plates to a subconfluent state, serum-starved for 24 hours and treated with. FI2K medium containing 10% fetal bovine serun (FBS), I 1 Ci/nil 3 H]-thymidine and 10 ng/ml basic Fibroblast Growth Factor (bFGF) (Gibco-BRL, Inc.) with various concentrations of Cyr61 and Fispl2 as indicated. NIH 3T3 fibroblasts were grown to subconfluence, serumstarved for 48 hours, and treated with Minimal Essential Medium (MEM) 81 containing 0.5% FBS, 1 C/Ci/ml 3 H]-thymidine, bFGF -and various concentrations of Cyr61 or Fispl2. Thymidine incorporation into the trichloroacetic acid-insoliible fractioi was determined after 24 hour incubation.

Logarithmically grown mink lung epithelial cells (Mvllu, CCL64) were treated with various concentrations of TGF-01 (Gibco-BRL) and 2 gg/ml of Cyr61 or Fispl2 for 18 hours; 3 H]-thynidine was then added to 1 gCi/nrl for 2 hours. Thymidine incorporation was determined as described above.

Purified recombinant Fispl2 protein did not exhibit any mitogenic activity under any tested assay conditions. Rather, Fispl2 was able 1 0 to enhance DNA synthesis induced by fibroblast growth factor in either NIH 3T3 fibroblasts or HUVE-cells. This activity was nearly indistinguishable from that exhibited by Cyr61.

Whereas in fibroblasts and endothelial cells, Cyr61 and Fispl2 enhance growth factor-induced DNA synthesis, both proteins can also enhance growth factor-mediated actions in another way. It is known that TGF-3 acts to inhibit DNA synthesis in epithelial cells (Satterwhite et al., 1994). It was observed that both Cyr61 and Fispl2 enhanced the ability of TGF-0 to inhibit DNA synthesis in mink lung epithelial cells. The data demonstrate that both recombinant Cyr61 and Fispl2, purified from senmm-free sources, are not mitogenic by themselves, but have the ability to synergize with the actions of polypeptide growth factors. Cyr61 and Fispl2 enhance DNA synthesis induction by FGF, and enhance DNA synthesis inhibition by TGF-0.

The present invention also comprehends the use of CTGF in methods to potentiate the mitogenic effect of true growth factors, or to screen for true growth factors. Those contemplated uses are in contrast to the reportedluse of CTGF as a mitogen or growth factor itself. U.S. Patent No.

5.408,040, column 7, line 65, to column 11, line 7, incorporated herein by reference hereinabove.

Further, the invention comprehends methods of screening for modulators of mitogen potentiation. A comparative assay exposes 82 subconfluent. cells to an ECM signalling .molecule such as Cyr61, a growth factor, and a suspected modulator of an ECM signalling molecule. As a control, similar cells are exposed to the ECM signalling molecule and the growth factor. A further control exposes similar cells to the growth factor and the suspected modulator in the absence of the ECM signalling molecule.

Based on the relative cell proliferation rates, as measured by, thymidine incorporation, an identification of a suspected modulator as a promoter of mitogen potentiation (elevated cell proliferation in the presence of all three molecules) or an inhibitor of mitogen potentiation (decreased cell S 10 proliferation in the presence of the three molecules) can be made.

Example 19 Cornea Assay For Angiogenic Factors And Modulators Another assay for modulators of angiogenesis is an in vivo assay for assessing the effect of a suspected modulator in the presence of an ECM signalling molecule-related biomaterial, such as Cyr61, on angiogenesis is the Cornea Assay. The Cornea Assay takes advantage of the absence of blood vessels in the cornea, which in the presence of an angiogenic factor, results in the detectable development of capillaries extending from the sclera into the cornea.. Friedlander et al., Science 270:1500-1502 (1995). This ingrowth of new blood vessels from the sclera can be microscopically monitored. Further, the visually determined rate of migration can be used to assess changes in the rate of angiogenesis. These cornea assays may be performed using a wide variety of animal models. Preferably, the cornea assays are performed using rats. By way of example, an assay for suspected modulators of Cyr61 using this assay is disclosed. To perform this assay, Cyr61 is initially titrated using primary capillary endothelial cells to determine effective concentrations of Cyr61. Subsequently, Cyr61, in the presence or absence of a suspected modulator, is surgically implanted into the corneas of mammalian laboratory animals. rabbits or rats. In a preferred embodiment, Cyr61 (or Cyr61 and a suspected modulator) is embedded in a biocompatible matrix, using 83 matrix materials and techniques that are standard in the art. Subsequently, eyes containing implants are visually observed for growth of the readily visible blood vessels within the eye. Control implantations may consist of physiologically balanced buffers embedded in the same type of matrix and implanted into eyes of the same type of laboratory animal receiving the Cyr61-containing implants.

The development of an in vivo cornea assay for angiogenic factors has advantages over existing in vitro assays for these factors. The process of angiogenesis involves four distinct phases: induction of vascular 10 discontinuity, endothelial cell movement, endothelial cell proliferation, and three-dimensional restructuring and sprouting. In vitro assays can evaluate only two of these steps: endothelial cell migration and mitogenesis. Thus, to provide a comprehensive assay for angiogenic factors, an in vivo assay such as the cornea assay is preferred.

The cornea assay has been used to confirm the effect of angiogenic factors such as Cyr61, Fispl2, CTGF, and Nov, on the process of angiogenesis. Moreover, modifying the cornea assay by including any of these angiogenic factors and a suspected.modulator of their activity results in a cornea assay for modulators of angiogenesis. For example, in one embodiment of the invention, dose of an angiogenic factor such as Cyr61 could be used in cornea assays for positive effectors of the angiogenic activity of Cyr61. An appropriate dose of Cyr61 would initially be determined by titration of the dose response relationship of Cyr6l with angiogenic events.

Inclusion of a control assay lacking Cyr61 would eliminate compounds having a direct effect on angiogenesis. In an alternative embodiment of the invention, an effective dose of an angiogenic factor such as Cyr6l could be used to assay for negative modulators of the activity of an angiogenic factor. In yet another alternative embodiment, a corneal implant comprises Cyr61 and another corneal implant comprises Cyr61 and a suspected modulator of angiogenesis.

Measurements of the development of blood vessels in the implanted corneas 84provides a basis for identifying a suspected modulator as a promoter of angiogenesis (elevated blood vessel development in the cornea containing an implant comprising the -suspected modulator. A relative decrease-in -blood vessel development identifies an inhibitor of angiogenesis.

The rat is preferred as the animal model for the cornea assay.

Disclosures in the art have established the rat model as a well-characterized system for analyzing angiogenesis. Parameters such as implant size, protein release dynamics, and suitable surgical techniques, have been well characterized. Although any strain of rat can be used in the cornea assay, 10 preferred strains will be well-characterized laboratory strains such as the Sprague-Dawley strain.

O Although rats of various sizes can be used in the cornea assay, a preferred size for the rats is 150-200 g/animal. Anesthesia is induced with Methoxyflurane and is maintained for 40-60 minutes with sodium pentobarbital (50 mg/kg, delivered intraperitoneally). The eyes are gently opened and secured in place by clamping the upper eyelid with a nontraumatic hemostat. Two drops of sterile proparacaine hydrochloride are then placed on each eye as to effect local anesthesia. Using a suitable surgical blade such as a No. 11 Bard Parker blade, an approximately 1.5 mm incision is made approximately 1 mm from the center of the cornea. The incision extends into the stroma but not through it. A curved iris spatula approximately 1.5 mm in width and approximately 5 mm in length is then inserted under the lip of the incision and gently blunt-dissected through the stroma toward the outer canthus of the eye. Slight finger pressure against the.

globe of the eye helps to steady the eye during dissection. The spatula penetrates the stroma no more than approximately 2.5 mm. Once the cornea pocket is made, the spatula is removed and the distance between the limbus and base of the pocket is measured to make sure the separation is at-least about I mm.

85 To provide slow release of the protein after implantation in the cornea, protein is mixed with poly-2-hydroxyethylmethacrylate (Hydron^), or an equivalent agent, to form a pellet of approximately 5 Implants made in this way are rehydrated with a drop of sterile lactated Ringers solution and implanted as described above. After implantation, the corneal pocket is sealed with erythromycin ointment. After implantation, the protein-Hydron pellet should remain near the limbus of the cornea (cornea-sclera border) and vision should not be significantly impaired.

i Following surgery, animals are examined daily for seven days with the aid of a stereomicroscope to check for inflammation and responses.

i-" To facilitate examination, the animal is anesthetized with Methoxyflurane and the anesthetic is continuously administered by nose cone during examination.

During this seven day period, animals are monitored for implant position and corneal exudate. Animals exhibiting corneal exudate are sacrificed. A preferred method of euthanasia is exsanguination. Animals are initially anesthetized with sodium pentobarbital (50 mg/kg) and then perfused, as described below.

After seven days, animals are perfused with colloidal carbon Ink). Anesthesia is induced with Methoxyflurane, and is maintained with sodium pentobarbital (50 mg/kg, intraperitoneally). Each animal is perfused with 100-200 ml warm (37 C) lactated Ringers solution per 150 g of body mass via the abdominal aorta. Once the snout of the animal is completely blanched, 20-25 ml of colloidal carbon is injected in the same way as the Ringers solution, until the head and thoracic organs are completely black. Eyes are then enucleated and fixed. Corneas are excised, flattened, and photographed.

Each protein is typically tested in three doses, in accordance with the practice in the art. Those of ordinary skill in the art realize that six positive corneal responses per dose are required to support an identification of an angiogenic response. An exemplary cornea assay includes three doses 86of the protein under study, with six rats being tested at each dose.

Additionally, six animals are exposed to a buffer-Hydron implant and serve as negative controls. Exposure -of at least three animals to a known angiogenic factor-Hydron implant serve as positive controls. Finally, to demonstrate the specificity of any observed response, six animals are exposed to implants containing a single dose of the protein under study, an excess of neutralizing antibody, and Hydron.

A cornea assay as described above was performred to assess the ability of Cyr61 to induce angiogenesis. Four animals were given negative 10 control implants containing a buffer-Hydron pellet (both eyes). Each of these :animals failed to show any blood vessel development in either eye after seven days. Six animals received implants containing a biologically effective amount of Fibroblast Growth Factor 15 in one eye and a control pellet in the other eye; all six showed angiogenic development in the eye receiving FGF, none showed neovascularization in the eye receiving the negative control.

Seven animals received 1 ug/ml Cyr61, in one eye and all seven of these eyes showed blood vessel growth; one of the seven eyes receiving a negative control showed angiogenic development. Finally, four animals received implants locally releasing 1 /,g/ml Cyr61 (Hydron prepared with a 10 pg/ml Cyr61 solution) and a specific anti-Cyr61 antibody in three-fold excess over Cyr61; none of the eyes of this group showed any angiogenic development.

Thus, the in vivo assay for angiogenesis identifies angiogenic factors such as FGF and Cyr61. The assay also is able to reveal inhibition of angiogenic development induced ECM signalling molecules such as Cyr61.

Example Blood Clotting ECM signalling molecules are also useful in correcting hemostasis, or abnormal blood clotting. A defect in blood clotting caused by, low level expression of cyr61 which thereby allows Tissue Factor 87 Pathway Inhibitor (IFPI) to act unchecked .can be corrected by expression.or use of recombinant Cyr61 protein.

Cyr6 can interact with TFPI, a protein that inhibits extrinsic blood coagulation. TFPI inhibits blood clotting in a two step process. First, TFPI binds to factor Xa and the TFPI:Xa .complex .then interacts with the Tissue Factor (TF):Factor VIIa complex, thereby inhibiting the latter complex.

The TF:Factor VIIa complex is the complex that activates factors IX and X.

By inhibiting TF:VIIa, TFPI regulates coagulation by preventing the activation of Factors IX and X, required for blood clotting. The interaction of Cyr61 10 with TFPI inhibits the activity of TFPI, thus promoting blood coagulation.

Cyr61 is, thus, a tissue factor agonist.

Because of the interaction of Cyr61 and TFPI, Cyr61 can control the ability of TFPI to inhibit coagulation, thereby regulating hemostasis. A defect in Cyr61 may lead to the inability to inhibit TFPI at the appropriate time, resulting in excessive inhibition of tissue factor, thereby preventing clot formation. Deregulated expression of Cyr61 will conversely inhibit the activity of TFPI constitutively, and thus tissue factor is constantly active, resulting in excessive clotting. When the expression of cyr61 in endothelial cells is deregulated, one possible outcome is thrombosis.

In addition to Cyr61, other ECM signalling molecules, such as Fispl2 and CTGF, have been shown to exert effects on cells participating in angiogenesis. Consequently, it is anticipated that a variety of ECM signalling molecule-related biomaterials, alone or in combination, may be used in the methods of the invention directed towards modulating hemostasis.

Example 21 Ex vivo Hematopoietic Stem Cell-Ciltures To investigate the effect of Cyr61 on the growth of primitive multipotent stem cells, several assays that distinguish these cells from more mature progenitor cells in a hematopoietic culture are employed. These assays make use of physicochemical (fibronectin-binding) or growth and 88 development-related (generation of progenitor blast colonies) differences between immature and mature subsets of cells.

Two cell lines-which require conditioned media for growth are used as a source of hematopoietic stem cells (HSC). These cloned, factordependent murine lines are B6Sut (cloned from long term bone marrow culture and capable of growing in liquid medium without differentiation, but multipotent in agar, as described in Greenberger et al., Proc. Natl. Acad. Sci.

[USA] 80:2931 [1983]), and FDCP-mix (cloned from long term bone marrow culture cells infected with the recombinant virus src-MoMuLV, and are 10 multipotent in agar cultures, as described in Spooncer et al., Nature 310:2288 [1984]). B6Sut cells are propagated in Kincaid's medium with 10%. fetal calf serum (FCS) and 10% 6X-concentrated WEHI-conditioned medium.

Greenberger et al. FDCP-mix cells are propagated in Fischer's medium with S. 20% horse serum and 10% 6X-concentrated WEHI-conditioned medium. The cell lines are propagated at 37 0 C, 5% CO,.

Various ex vivo or in vitro cultures are assayed for population growth in the presence or absence of exogenously supplied murine Cyr61 or polyclonal anti-Cyr61 antibodies. Under limiting dilution conditions, the cobblestone area forming cell (CAFC) assay is used to identify cells with long tenn repopulating ability. Ploemacher et al., Blood 74:2755 (1989); Ploemacher et al., Blood 78:2527 (1991). Cells identified as having long term repopulating ability by the CAFC assay are then analyzed by measuring three parameters: Rate of population doubling, mitotic index, and rate of DNA synthesis.

Long term cultures, with or without supplementation with Cyr61, are assayed for their levels of primitive HSC in-the-CAFC assay--van der Sluijs et al., Exp. Hematol. 22:1236 (1994). For example, M2-10B4 stromal cells, B6Sut, and FDCP-mix are each sujected to the CAFC assay in the following manner, described for the M2-10B4 cell line. Stromal cell layers are prepared by inoculating 5 x 10 M2-10B4 stromal cells (a cell line 89 cloned from bone marrow -stroma, Sutherland et-al., Blood 78:666 [1991]-) into each well of a 96-well culture plate in DMEM with 10% FCS. When the cells approach confluency, they are rinsed with PBS and irradiated (20 Gy of gamma-irradiation, 1.02-1.04 Gy/minute) to prevent replication of any hematopoietic cells within the stroma, without affecting -the stroma's ability to support hematopoiesis.

Hematopoietic stem cells are added to the irradiated stromal cells in DMEM with 10% FCS, in the presence or absence of Cyr61 g/ml final concentration). Population doubling rates are detennined, e.g., 10 by microscopic examination of cell morphology to determine the numbers of long term repopulating cells (and more mature short term progenitor cells) present in the various experimental long term cultures. Subsequent investigation of the expansion and differentiation capacities of the potential long term HSC cultures is used for confirmation of suitable candidate cell lines.

The mitotic index is determined according to procedures standard in the art. Keram et al., Cancer Genet. Cytogener. 55:235 (1991).

Harvested cells are fixed in methanol:acetic acid counted, and resuspended at 106 cells/ml in fixative. Ten microliters of this suspension is placed on a slide, dried, and treated with Giemsa stain. The cells in metaphase are counted under a light microscope, and the mitotic index is calculated by dividing the number of metaphase cells by the total number of cells on the slide. Statistical analysis of comparisons of mitotic indices is performed using the 2-sided paired t-test.

The rate of DNA synthesis is measured using a thymidine incorporation assay. Various cultures are propagated in l1Ci/ml thymidine (ICN Biomedicals, Inc., Costa Mesa, CA) for 24-72 hours.

Harvested cells are then rinsed with PBS and fixed with 10% trichloroacetic acid. DNA is dissolved in 0.1 N NaOH, and thymidine incorporation is detennined, for example by liquid scintillation spectrophotometry.

The use of an ECM signalling molecule-related biomaterial, such as Cyr61, can be used in the ex vivo expansion of hematopoietic stem cell cultures. In addition, more than one ECM signalling molecule-related biomaterial may be used to expand these cultures. For example, Cyr61, with 5 its expression targeted locally, may be combined with Fispl2, which exhibits a more expansive targeting as evidenced by the presence of Fispl2 in culture media. As an alternative, CTGF may be substituted for Fispl2, its mouse ortholog. One of skill in the art would be able to devise other combinations of ECM signalling molecule-related biomolecules that are within the spirit of S 10 the invention.

Those of ordinary skill in the art will recognize that the successful expansion of hematopoietic stem cell cultures in the presence of ECM signalling molecules such as Cyr61 provides a basis for a method of screening for suspected modulators of that expansion process. As in the other methods of the invention, a suspected modulator is combined with an ECM signalling molecule such as Cyr61 and exposed to primitive cells. In parallel, the ECM signalling molecule is exposed to similar cells. The relative rates of expansion may be used to identify a promoter, or inhibitor, of the ability of the ECM signalling molecule to expand pluripotent hematopoietic stem cell cultures.

Cyr61, alone or in combination with other hematopoietic growth factors, may also be used to expand stem cell populations taken from a patient and which may, after expansion, be returned to the patient or other suitable recipient patient after for example, chemotherapy or other treatment modalities that result in the depletion of blood cells in a patient. Stem cell populations expanded according to the present invention may also be used in bone marrow transplants in a patient in need thereof.

91 Example 22 Organ regeneration The role of Cyr61 in the various cellular processes invoked by changes in the cellular growth state indicate that this protein would be effective in promoting organ regeneration. Towards that end, studies were conducted to determine the expression profile of murine cyr61 in remaining liver tissue following a partial hepatectomy. (The response of remaining liver tissue following partial hepatectomy is a model for the liver's response to a S variety of injuries, including chemical injuries, exposure to toxic levels 10 of carbon tetrachloride.) BALB/c 3T3 (Charles River) mice were subjected to partial hepatectomies removing approximately 67% of their liver tissue. Higgins et al., Archs. Path. 12:186-202 (1931). Twenty microgram aliquots of RNA were removed from the remaining liver tissue at varying times following the operation and liver RNA was isolated by tissue homogenization followed by guanidinium isothiocyanate, cesium chloride precipitation. Sambrook et al.

RNAs were then immobilized on nitrocellulose filters and probed with radiolabeled clones containing various regions of murine cyr61 cDNA.

Results were visualized by autoradiography and indicated that removal of liver 20 tissue induced cyr61 mRNA expression, particularly in cells found near the injury site. Consequently, induction of cyr61 expression, by recombinant techniques, might promote the regeneration of organs such as liver. For example, cyr61 expression can be controlled, by introducing recombinant cyr61 constnucts that have been engineered to provide the capacity to control expression of the gene, by the use of tissue-specific promoters, the K14 promoter for expression in skin. The recombinant cyr61 may be introduced to cells of the relevant organ by gene therapy techniques using vectors that facilitate homologous recombination vectors derived from Herpesvinises, Adenovinrs, Adeno-associated Virus, Cytomegalovirus, Baculovirus, retroviruses, Vaccinia Virus, and others).

92 Techniques for introducing heterologous genes into eukaryotic cells, and techniques for integrating heterologous genes into host chromosomes by homologous recombination,-are -well-known -in -the -art.

The development of skin, another organ, is also affected by Cyr61. The expression of cyr61 is induced in cells in the vicinity of skin injuries. Also, as described above, Cyr61 has a chemotactic effect Cyr61 induces cell migration) on endothelial cells and fibroblasts. Further, Cyr61 induces the proliferation of endothelial cells and fibroblasts. Both processes are involved in the healing of skin wounds. Accordingly, Cyr61 10 administration, by localized or topical delivery, should promote skin regeneration.

:Cyr61 is also highly expressed in lung epithelium. These cells are frequently injured by exposure to environmental contaminants. In particular, lung epithelium is frequently damaged by-air-borne oxidants. The 15 administration of Cyr61, in atomizers or inhalers, may contribute to the healing of lung epithelium damaged, by environmental contaminants.

Example 23 SChondrogenesis- ECM Signalling Molecules Are Expressed in Mesenchyme 20 Some ECM signalling molecules are also expressed in cells, such as mesenchyme cells, that ultimately become a part of the skeletal system. In this Example, Cyr61 is identified as one of the ECM signalling molecules expressed in mesenchyme cells. Limb mesenchymal cells were grown in micromass culture as described above on glass coverslips (Fisher) for 3 days. Cultures were fixed in 4% parafonnaldehyde in PBS, incubated for 30 minutes at room temperature with I mg/ml bovine testicular hyaluronidase (type IV, Sigma) in 0. IN sodium acetate (pH 5.5) with protease inhibitors phenymethylsulfonyl fluoride (PMSF, 1 mM), pepstatin (1 pg/ml), leupeptin (1 Ag/ml), aprotinin (1 g.g/ml), aminocaproic acid (50 mM), benzamidine (5 mM), and EDTA (1 mM), blocked with 10% goat serum in 93 PBS and incubated overnight at 4"C -with primary antibodies against Cyr61 (Yang et al., 1991), fibronectin (Gibco) and tenascin (Gibco). Controls were incubated with anti-Cyr61 antibodies neutralized with I ig/ml purified Cyr61.

Cultures were subsequently incubated with FITC-conjugated goat-anti-rabbit secondary antibody (Zymed), for-1 -hour -at room -temperature.

For whole mount immunohistochemical staining, mouse embryos from gestational days 10.5 to 12.5 were fixed in 4% paraformaldehyde in PBS, dehydrated in methanol/PBS and stored at -20"C in absolute methanol. After rehydration, embryos were incubated with antio 10 Cyr61 antibodies as described in Hogan et al., Development 120:53-60 (1994), incorporated herein by reference. Controls were incubated with anti- Cyr61 antibodies neutralized with 1 pg/ml purified Cyr61. Immunostained embryos were fixed, cleared and photographed.

Consistent with the transient expression of the cyr61 mRNA in somitic mesenchymal cells that are differentiating into chondrocytes (O'Brien et al., 1992), the Cyr61 protein was found in the developing embryonic skeletal system. Cyr61 was localized by whole mount immunohistochemical staining to the proximal limb bud mesenchyme in gestational day 10.5 to 12.5 embryos. The Cyr61 protein was localized to the developing vertebrae, the 20 calvarial frontal bone and the first brachial arch, as well as in the heart and umbilical vessels, forming an expression pattern that was consistent with the cyr61 mRNA expression pattern (O'Brien et al., 1992).

Cyr61 protein could be detected by immunoblot analysis in whole limb buds and in micromass cultures of limb bud mesenchymal cells.

The level of Cyr61 protein remained at relatively constant levels throughout the 4 day culture period during which chondrogenesis occurred.. Using quantitative immunoblot analysis, Cyr61 was estimated to represent approximately 0.03% of total cellular and extracellular proteins in the mesenchymal cell cultures. Cyr61, tenascin (Gibco), and fibronectin were localized to the cartilage nodules by immunofluorescent staining in the -94mesenchymal cell cultures.. Cyr61 and tenascin were primarily localized among the intranodular cells, while a fibrillar staining pattern was also observed around and between-the cartilage nodules with anti-fibronectin antibodies. A similar immunofluorescent staining pattern was observed in transverse sections of the micromass cultures for all three antibodies.

Together, these results show that endogenous Cyr61 is localized in, the developing limb bud mesenchyme, both in vivo and in vivo.

Example 24 Chondrogenesis- ECM Signalling Molecules Promote Cell Adhesion 1 0 Cyr61 is a secreted protein that mediates the adhesion of fibroblasts and endothelial cells to non-tissue culture-treated plastic surfaces (Kireeva et al., Mol. Cell. Biol. 16.1326-1334 [1996]). The attachment of limb bud mesenchymal cells on non-tissue culture dishes coated with BSA, Cyr61, tenascin, and fibronectin, were compared.

Cyr61, fibronectin (Gibco), or tenascin (Gibco) were diluted in 0.1 protease-free bovine serum albumin (BSA) in PBS with 0.5 mM PMSF, to a final concentrations of 10 or 50 pg/ml. A 10 /l drop/well was placed in a non-tissue culture treated 24-well plate (Coming), and incubated at room temperature for 2 hours. The wells were blocked with 1% BSA in PBS for 1 hour at room temperature, and rinsed with serum-free MEM (Modified Eagle's Medium). Limb mesenchymal cells, suspended at 5 x 105 cell/ml in serum-free MEM, were added at a volume of 400 pl/well, and incubated at 37C, 5 CO, for 1 or 3 hours. At each time point, the cell suspension was removed, the wells were rinsed with MEM and the remaining adherent cells were photographed.

Cells attached poorly to BSA-coated dishes, but adhered as ,clusters of rounded cells to Cyr61- and tenascin-coated dishes within 1 hour of plating. In contrast, cells plated on fibronectin-coated dishes attached unifomnly and started to spread. When cells were allowed to attach for 3 95 hours, many -more adherent cells-were observed. Furthermore, intercellular clustering and rounded cell morphology were maintained in cells plated on Cyr6I and tenascin, while cells plated on fibronectin spread to form a monolayer. These observations show that Cyr61 mediates the adhesion and maintenance of a -rounded cellular morphology which is conducive for mesenchymal cell chondrogenesis (Zanetti et al., Dev. Biol. 139:383-395 [1990]; Solursh et al., Dev. Biol. 94.259-264 [1982]), similar to that previously reported for tenascin (Mackie er al., J. Cell Biol. 105:2569-2579 [1987]).

10 As mentioned previously, ECM signalling molecules such as Cyr61 may be used in methods of screening for modulators of cell adhesion, including, but not limited to, the adhesion of chondrocytes. The comparative assay, described above, measures the relative adhesion levels of cells exposed to a combination of an ECM signalling molecule and a suspected modulator 15 of cell adhesion and cells exposed to the ECM signalling molecule alone, whereby the relative levels provide a basis for identifying either a promoter or an inhibitor of cell adhesion.

Example Chondrogenesis- ECM Signalling Molecules Promote Cell Aggregation Since aggregation is an essential step for chondrogenic differentiation (Solursh, In The role of extracellular matrix in development, pp. 277-303 (Trelstad, ed.) (Alan R. Liss, New York 1984)), the ability of Cyr61 to mediate intercellular aggregation in suspension cultures of mesenchymal cells was assessed. The number of cells remaining at various times after isolation were counted. Untreated mesenchymal cells in suspension began to aggregate soon after isolation, as the number of single cells was decreased to 30% of the initial number within a 2 hour incubation period. Cell aggregation was significantly inhibited in cultures treated with affinity-purified anti-Cyr61 antibodies, indicating that endogenous Cyr61 is -96 important for mesenchymal cell aggregation. To rule out..the possibility, that the affinity-purified anti-Cyr61 antibodies might contain undefined components that interfere with aggregation, anti-Cyr61 antibodies, described above, were pre-incubated with purified Cyr61 protein prior to addition to cells. These pre-incubated antibodies affected cell aggregation no more than the IgG and Cyr61 buffer controls, indicating that the anti-Cyr61 antibodies achieved their inhibition of cell aggregation by neutralizing the endogenous Cyr61 protein of mesenchymal cells.

In addition to the cell aggregation in suspension cultures 10 described above, the effect of Cyr61 on mesenchymal cell aggregation in micromass cultures was also examined. When purified Cyr61 protein (0.3 g/ml) was added to limb mesenchymal cells, precocious cellular aggregation was observed within 24 hours, unlike control cells which had not received Cyr61. Neither Cyr61-treated nor control cultures had differentiated into 15 cartilage nodules at this time. By culture day 3, the development of interodular cellular condensations between the distinct cartilage nodules was more extensive in Cyr61-treated cultures. These internodular condensations subsequently undergo chondrogenesis, observed as Alcian, blue-staining cartilaginous matrix on culture day 4. Taken together, these results indicate 20 that Cyr61 is able to promote cell-cell aggregation, a necessary step in chondrogenesis of mesenchymal cells in micromass culture.

Example 26 Chondrogenesis- ECM Signalling Molecules Promote Cell Proliferation Some ECM signalling molecules, such as Cyr61, affect chondrogenesis, as revealed by effects on limb bud mesenchyme cells in micromass culture, as described above. Ahrens et al., Dev. Biol. 60:69-82 (1977), has reported that these cells, in micromass culture, undergo chondrogenesis in a manner similar to the in vivo process. Mesenchyme cells were obtained from mouse embryonic limb buds by trypsin digestion (1 -97 mg/ml, 1:250 dilution of-porcine pancreatic trypsin, Sigma Chemical Co.).

Cells were explanted in plastic tissue culture wells and allowed to attach for 2 hours at 37°C, 5% CO,. Cells were then incubated for 24 hours at 37°C, CO, in MEM with 10% FBS, penicillin (50 U/ml), and streptomycin /Ag/ml). At this point, the composition of the medium was changed by substituting 4% NuSerum (Collaborative Biomedical Products, Inc.) for FBS. Individual cultures then received Cyr61, fibronectin, heparin, (each at approximately I gg/ml) or buffer as a negative control. An additional control was provided by adding a 1:100 dilution of affinity-purified anti-Cyr61 .i. 10 antibody (approximately 13 xg/ml stock solution), elicited and purified by standard techniques. Harlow et al.

Cell proliferation was assessed by the thymidine assay, described above, and by microscopic cell counts. Chondrogenesis was assessed by quantifying the incorporation of 35 S]-sulfate (ICN Biomedicals, 15 Inc.) into sulfated glycosaminoglycans, and by qualitatively determining the extent of chondrogenesis by cell staining with Alcian Blue. Cultures, described above, were labeled with 2.5 ACi/ml 3 "S]-sulfate for 18 hours, washed twice in PBS, fixed with Kahle's fixative (Pepper et al., J. Cell Sci.

109:73-83 [1995]) and stained for 18 hours in 0.5% Alcian Blue, pH 20 The extent of chondrogenesis is correlated with the intensity of Alcian Blue staining. San Antonio et al., Dev. Biol. 115:313-324 (1986). The results show that Cyr6 specifically increased limb bud mesenchyme cell proliferation and aggregation, leading to enhanced chondrogenesis.

In addition to demonstrating that purified Cyr61 enhanced growth factor-induced DNA synthesis in fibroblasts and endothelial cells, the effects of Cyr61 on cell proliferation were directly examined. Cell proliferation during the 4 day culture period was determined by counting cell number and by incorporation of ['H]-thymidine. To determine cell number, cells were harvested by trypsin/EDTA (Sigma) and counted with a Coulter counter. In parallel cultures, 3 H]-thymidine (1 gCi/ml; ICN) was added to 98 the media for 18 hours and incorporation in .the TCA-insoluble layer was determined by liquid scintillation counting. Purified Cyr61 protein added to limb mesenchymal cells both increased cell number and enhanced DNA synthesis after 2 and 3 days in culture, although the total cell number in Cyr61-treated and Cyr61-untreated cultures leveled off at the same level after 4 days.

The role of Cyr61 in chondrogenesis may also improve the integration of prosthetic devices. For example, skeletal injuries and conditions frequently are treated by the introduction of a prosthesis hip prosthesis, 10 knee prosthesis. Beyond questions of histocompatibility, the successful implantation of a prosthetic device requires that the foreign element become integrated into the organism's skeletal structure. The capacity of Cyr61 polypeptides to affect cell adhesion, migration, and proliferation, and the ability of Cyr61 polypeptides to induce the differentiation of mesenchyme cells S 15 into chondrocytes, should prove valuable in the treatment of skeletal disorders by prosthesis implantation. For example, integration of a prosthetic device by chondrocyte colonization would be promoted by therapeutic treatments involving the administration of Cyr61 in a pharmaceutically acceptable adjuvant, carrier or diluent, using any of the administration routes known in 20 the art or by coating the prosthesis device with Cyr61 polypeptides in a suitable carrier. The carrier may also be a slow-release type vehicle to allow sustained release of the polypeptides.

As noted in previously, the methods of the invention include a method of screening for modulators of cell proliferation, including chondrocytes. A comparison of the relative rates of cell proliferation in the presence of a control comprising an ECM signalling molecule alone Cyr61) and in the presence of a combination of an ECM signalling molecule and a suspected modulator of cell proliferation provides a basis for identifying a suspected modulator as a promoter, or inhibitor, of chondrocyte proliferation.

99 Example 27 Chondrogenesis- ECM Signalling MoleculesPromote Chondrogenesis Chondrogenic differentiation was quantitated by incorporation of 3 S]-sulfate (ICN) into sulfated glycosaminoglycans and assessed qualitatively by Alcian Blue staining. Cultures were radiolabeled with /Ci/ml ["S]-sulfate for 18 hr, fixed with Kahle's fixative and stained with Alcian Blue, pH 1.0 (Lev et al., 1964). The extent of chondrogenesis is correlated with the intensity of Alcian Blue staining (San Antonio et al., 1986). 5 S]-Sulfate incorporation in the fixed cell layer was quantitated by 10 liquid scintillation counting.

Exogenous purified Cyr61 protein promoted limb mesenchymal cell aggregation and resulted in greater Alcian blue-staining cartilaginous regions in micromass cultures, suggestive of a chondrogenesis-promoting effect. This effect was quantified by the incorporation of ["S]-sulfate into 15 sulfated glycosaminoglycans (San Antonio et al., 1986) in Cyr61-treated micromass cultures. Exogenous Cyr61 enhanced ["S]-sulfate incorporation in a dose-dependent manner, resulting in a 1.5-fold and 3.5-fold increase with 0.3 and 5 p/g/ml Cyr61, respectively, and was correlated qualitatively by increased Alcian Blue staining. The increase observed at the 5 /ug/ml Cyr61 dose (120 nM) is an under-estimation of the actual extent of chondrogenesis, since some of the large cartilage nodules which were formed at this dose detached from the dish. Cultures treated with 10 /g/ml Cyr61 formed a more massive mound of cartilage.

A review of the literature indicated that chondrogenesis in limb mesenchymal cell micromass cultures was increased 2-fold with the addition of 10 Ag/ml heparin (San Antonio et al., 1987; Resh et al., 1985) and 3-fold with 50 Ag/ml tenascin (200 nM) (Mackie et al., 1987). The results demonstrated that purified Cyr61 was effective at concentrations (10-100 nM) similar to or less than those of other molecules known to promote chondrogenesis in this cell system.

100 Since a certain threshold cell density mustbe reached for initial aggregation to occur (Umansky, 1966; Ahrens er al., 1977), embryonic mesenchymal cells plated at low densities are normally unable to differentiate into chondrocytes, although the addition of exogenous factors such as heparin or poly-L-lysine (San Antonio er al., 1986; San Antonio et al., 1987) have been shown to support chondrogenesis in cells plated under these conditions.

Therefore, the ability of Cyr61 to promote differentiation of mesenchymal cells plated at densities above and below the threshold for chondrogenesis was assessed. Cells plated at 2.5 x 106 cell/ml incorporated little 35 S]-sulfate.

10 However, when Cyr61 was added, these sub-threshold density cultures formed nodules and incorporated sulfate to a level similar to that in cultures plated at a.

3 x 10 cells/ml, which supports chondrogenesis. Therefore, Cyr61 can promote chondrogenesis in mesenchymal cells plated at non-chondrogenic, sub-threshold densities.

15 It is conceivable that when mesenchymal cells are plated in a high density micromass, the extent of chondrogenesis may be maximal and cannot be enhanced further by exogenous factors, which also may not be accessible to all cells. However, addition of exogenous Cyr61 resulted in a 2-fold enhancement in 3 5 ]-sulfate incorporation in cultures plated at densities ranging from 3 to 10 x 106 cell/ml. Therefore, Cyr61 can further enhance chondrogenesis in high density micromass cultures, which have apparently not reached a maximal degree of differentiation.

It is possible that the increased ["S]-sulfate incorporation in Cyr61-treated cultures is at least partly due to an increase in cell number, since Cyr61 also promotes cell proliferation. If this were true, then normalization of sulfate- incorporation with respect to cell number would eliminate any differences between control and Cyr61-treated cultures. This was not found to be the case. Cyr61-treated cultures still showed an approximately 2-fold increase in normalized sulfate incorporation over control, indicating that Cyr61 promotes a net increase in chondrogenesis. On culture 101 day 2, the sulfate/cell number ratio was lower in Cyr61-treated cultures compared to controls and is reflective of a low level of ["S]-sulfate incorporation relative to cell number, since mesenchymal cells are mostly proliferating rather than differentiating in these early stage cultures (Ede, 1983).

The presence of endogenous Cyr61 in these cells, both invivo and in vitro, indicates-that Cyr61 may indeed function biologically to regulatechondrogenic differentiation. The ability of exogenously added purified Cyr61 to promote intercellular aggregation and to increase ["S]-sulfate incorporation 10 and Alcian-blue staining in limb mesenchymal cells demonstrates that Cyr61 can act as a chondrogenesis enhancing factor. As shown above in Example 11, anti-Cyr61 antibodies can neutralize both the cell adhesion and DNAsynthesis enhancement activities of Cyr61. Anti-Cyr61 antibodies were added to the mesenchymal cell culture media or mixed the cell suspension prior to 5 plating. Chondrogenesis was inhibited in the cultures treated with anti-Cyr61 antibodies, as demonstrated by decreases of ["S]-sulfate incorporation to and 30% of controls, when antibodies were added to the media, and mixed with the cells, respectively. These observations were correlated with decreased Alcian Blue staining. However, mixing of the anti-Cyr61 antibodies 20 with mesenchymal cells prior to plating resulted in complete detachment in some of the treated cultures within 24 hours.

To eliminate the possibility of an unidentified component in the antibody preparation as a cause of cell detachment, anti-Cyr61 antibody was preincubated with 1 pg/ml purified Cyr61 protein prior to mixing with cells.

The inhibition of chondrogenesis in mesenchymal cells mixed with neutralized anti-Cyr61 antibodies was abolished.

Generally, the invention contemplates a method of screening for modulators of chondrogenesis. A comparative assay involves the exposure of chondrocytes to either a combination of a suspected nodulator of chondrogenesis and an ECM signalling molecule such as Cyr61, or the 102 ECM signalling molecule alone. Measurements of the relative rates of chondrogenesis then provide a basis for identifying the suspected modulator of chondrogenesis as a promoter or inhibitor of that process.

The results described in this Example demonstrate that endogenous Cyr61 is present in mesenchymal cells and is important for their chondrogenesis. Accordingly, the use of an ECM signalling molecule, such as Cyr61, to induce bone healing is contemplated by the invention. For example, a biologically effective amount of Cyr61 is introduced into a matrix such as a sponge, as described above, and this material is then applied to set 10 bone fractures or used to gather together the fragments of a comminuted bone fracture. A biodegradable matrix may be employed, or the matrix may be removed at an appropriate later time. Alternatively, Cyr61 may be applied e directly to bone. In addition, Cyr61 may be applied to inanimate objects such as biocompatible prosthesis, as described in Example 26.

Example 28 Genetics Another way to control the effects of an ECM signalling molecule-related biomaterial is to inactivate it by creating dominant negative mutations in the relevant gene in actively growing and dividing cells. One approach involves the use of recombinant techniques, to create homozygous mutant genotypes in ex vivo cultures such as HSC cultures.

Introduction of these cells into an organism, a human patient, would then provide an opportunity for the introduced mutant cells to expand and alter the expression of the ECM signalling molecule in vivo. Mutants homozygous for such a mutation could affect the expression of an enadogenous wild type ECM signalling molecule in other cells. Heterozygous mutants might produce altered ECM signalling molecules capable of interacting with the wild type ECM signalling molecule, also being expressed, in such a way that the ECM signalling molecule's activities are modulated or abolished.

103 Furthermore, because of the role played by ECM signalling molecules such as Cyr61 in regulating chondrogenesis skeletal development), genetic manipulations that alter the expression of human Cyr61 may prove medically important for prenatal screening methods and gene therapy treatments related to skeletal conditions, in addition to angiogenic conditions. For example, the cyr61 gene is expressed when mesenchymal cells of both ectodennal -and -mesodermal -origins differentiate -to form chondrocytes. Thus, one of the roles that Cyr61 might play is to regulate the commitment of mesenchyme cells to chondrocyte cell lineages involved in 10 skeletal development. Consistent with this view, transgenic mice overexpressing cyr61 ectopically are born with skeletal abnormalities. In all cases examined, the presence of the skeletal deformities correlates with expression of the transgene. These results suggest that the human form of Cyr61 may also regulate chondrogenesis and skeletal development. It is also possible that the human cyr61 gene may correspond to a genetic locus already known to affect skeletal development or birth defects relating to bone morphogenesis. Knowledge of the human Cyr61 protein sequence, presented in SEQ ID NO:4 herein, and the coding sequence of the cDNA, presented in ::SEQ ID NO:3 herein, provide the basis for the design of a variety of gene 20 therapy approaches.

This information also provides a basis for the design of probes useful in genotypic analyses, Restriction Fragment Length Polymorphism analyses. Such analyses are useful in the fields of genetic counselling, e.g., in diagnosing diseases and conditions and the likelihood of their occurrence, as well as in forensic analyses.

By way of example, the materials of the present invention are useful in the prenatal screening for a variety of conditions or disorders, including blood disorders, skeletal abnonnalities, and cancerous conditions.

Well known techniques for obtaining fetal cells, amniocentesis, provide the materials needed for diagnosis. In one embodiment of the invention, the 104fetal cells are expanded and DNA is isolated. In another embodiment, fetal cells are lysed and polymerase chain reactions are performed using oligonucleotide primers according to the invention. Using either approach, the DNA is then subjected to analysis. One analytical approach involves nucleotide sequence determination of particular regions of cyr6l or of the entire gene. The available human cyr61 coding sequence, presented in 'EQ ID NO:3 herein, facilitates the design of sequencing primers that brings nucleotide sequence analysis into the realm of practical reality. An alternative to nucleotide sequence analysis is an investigation of the expression 10 characteristics of the fetal nucleic acid. The capacity of the fetal nucleic-acid to be expressed might be dispositive in the diagnosis of Cyr61-related angiogenic, chondrogenic, or oncogenic disorders.

The invention also comprehends a kit comprising Cyr61. The i kits according to the invention provide Cyr61 in a form that is useful for 15 performing the aforementioned methods of the invention. Kits according to the invention contain isolated and purified recombinant human Cyr61 in a suitable buffer, optionally stabilized by the addition of glycerol for storage at In addition to the Cyr61 provided in the kit, the invention also contemplates the inclusion of any one of a variety of buffering agents, salts of various types and concentrations, and additional protein stabilizing agents such as DTT, all of which are well known in the art. Other kits according to the invention incorporate isolated and purified murine Cyr61. Kits incorporating a Cyr61 polypeptide and an inhibitory peptide or an anti-Cyr61 antibody, as described above, are also contemplated.

105 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Lau, Lester F.

(ii) TITLE OF INVENTION: Extracellular Matrix Signalling Molecules (iii) NUMBER OF SEQUENCES: 17 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Marshall, O'Toole, Gerstein, Murray Borun STREET: 6300 Sears Tower, 233 South Wacker Drive CITY: Chicago STATE: Illinois COUNTRY: United States of America ZIP: 60606-6402 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: Patentln Release Version #1.30 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(viii) ATTORNEY/AGENT

INFORMATION:

NAME: Clough, David W.

REGISTRATION NUMBER: 36,107 REFERENCE/DOCKET NUMBER: 28758/33766 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 312/474-6300 TELEFAX: 312/474-0448 TELEX: 25-3856 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 1480 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: CDS LOCATION: 180..1316 (ix) FEATURE: NAME/KEY: misc feature OTHER INFORMATION: "Mouse cyr61 cDNA coding sequence" (xi).SEQUENCE DESCRIPTION: SEQ ID NO:1: CGAGAGCGCC CCAGAGAAGC GCCTGCAATC TCTGCGCCTC CTCCGCCAGC ACCTCGAGAG AAGGACACCC GCCGCCTCGG CCCTCGCCTC ACCGCACTCC GGGCGCATTT GATCCCGCTG 120 106 CTCGCCGGCT TGTTGGTTCT GTGTCGCCGC GCTCGCCCCG GTTCCTCCTG CGCGCCACA ATG AGC TCC AGC Met Ser Ser Ser 1

ACC

Thr 5 TTC AGG ACG CTC GCT GTC GCC Phe Arg Thr Leu Ala Val Ala 10 GTC ACC CTT CTC Val Thr Leu Leu is CAC TTG ACC His Leu Thr CCT CTG GAG Pro Leu Giu AGA CTG GCG CTC TCC ACC Arg Leu Ala Leu Ser Thr 25 TGC CCC GCC GCC TGC CAC TGC Cys Pro Ala Ala Cys His Cys 275 323 GCA CCC AAG TGC Ala Pro Lys Cys CCG GGA GTC GGG Pro Gly Val Gly

TTG

Leu GTC CGG GAC Val Arg Asp' GGC TGC Gly Cys so GGC TGC TGT AAG Gly Cys Cys Lys

GTC

Val 55 TGC GCT AAA CAA CTC AAC GAG GAC TGC Cys Ala Lys Gin Leu Asn Giu Asp Cys

C

4 C CC* C.

C

C

AGC

Ser AAA ACT G-AG CCC Lys Thr Gin Pro

TGC

Cys 70 GAC CAC ACC AAG Asp His Thr Lys

GGG

Gly 75 TTG GAA TGC AAT Leu Giu Cys Asn TrC Phe GGC GCC AGC TCC Gly Ala Ser Ser

ACC

Thr 85 GCT CTG AAA GGG Ala Leu Lys Gly

ATC

Ile 90 TGC AGA GCT GAG Cys Arg Ala Gin TGA GAA Ser Glu 467 515 GGC AGA CCC Gly Arg Pro TTC GAG CCC Phe Gin Pro 115

TGT

Cys 100 GAA TAT AAC TCC Glu Tyr Asn Ser

AGA

Arg 105 ATC TAC CAA AAC Ile Tyr Gin Asn GGG GAA AGC Gly Giu Ser 110 GGC GCC GTG Gly Ala Val AAC TGT AAA CAC Asn Cys Lys His

GAG

Gin 120 TGC ACA TGT ATT Cys Thr Cys Ile

GAT

Asp 125 GGC TGC Gly Cys 130 ATT CCT CTG TGT Ile Pro Leu Cys

CCC

Pro 135 CAA GAA CTG TCT Gin Glu Leu Ser

CTC

Leu 140 CCC AAT CTG GGC Pro Asn Leu Gly

CC

S

C

TGT

Cys 145 CCC AAC CCC CGG Pro Asn.Pro Arg

CTG

Leu 150 GTG AAA GTC AGC Val Lys Val Ser

GGG

Gly 155 CAG TGC TGT GAA Gin Cys Cys Giu TGG GTT TGT GAT GAA Trp Val Cys Asp Glu 165 GAC AGC ATT AAG Asp Ser Ile Lys TCC CTG GAC GAC Ser Leu Asp Asp GAG GAT Gin Asp 175 GAC CTC CTC Asp Leu Leu GAG TTA ATC Giu Leu Ile 195

GGA

Gly 180 CTC GAT GCC TCG Leu Asp Ala Ser

GAG

Glu 185 GTG GAG TTA ACG Val Giu Leu Thr GGA ATT GGA AAA Ala Ile Gly Lys

GC

Gly 200 AGC TCA CTG AAG Ser Ser Leu Lys

AGG

Arg 205 AGA AAC AAT Arg Asn Asn 190 CTT CCT GTC Leu Pro Val GCC GAT GGC Ala His Gly TTT GGC Phe Gly 210 CAG AA Gin Lys 225 ACC GAA CCG CGA Thr Giu Pro Arg

GTT

Val 215 CTT 'ITC AAC CCT L-eu Phe Asn Pro CTG CAC Leu His 220 TGC ATC GTT GAG ACC ACG TCT TGG Cys 1-le Val Gin Thr Thr Ser Trp 230

TCC

Ser 235 GAG TGC TCC AAG AGC Gin Cys Ser Lys Ser TGC GGA ACT GGC ATC TCC AGA CGA GTI' ACC AAT GAC AAC CGA G-AG TGC 947 -107 Cys Gly Thr Gly Ile Ser Thr A-rg Val Thr Asa 245 250- Asp Asn Pro Glu Cys 255 CGC CTG GTG AAA GAG ACC CGG ATC TGT GAA GTG CGT CCT Arg Leu Val-.Lys 260 CCA GTG TAC AGC Pro Val Tyr Ser 275 .Glu Thr...Arg.11e -Cys 265 -G2.u -Va) Arg Pro TGT GGA CAA Cys Gly Gin 270 AAG ACC AAG Lys Thr Lys AGC CTA AAA Ser Leu Lys

AAG

Lys 280 GGC AAG AAA TGC Gly Lys Lys Cys

AGC

Ser 285 AAA TCC Lys Ser 290 CCA GAA CCA GTC Pro Glu Pro Val

AGA

Arg 295 TTr ACT TAT GCA Phe Thr Tyr Ala

GGA

Gly 300 TGC TCC AGT GTC Cys Ser Ser Val.

AAG

Lys 305 AAA TAC CGG CCC Lys Tyr Arg Pro AAA TAC TGC GGC TCC TGC GTA GAT GGC CGG TGC Lys Tyr Cys Gly Ser Cys Val- Asp Gly Arg Cys 310 1315 320 TGC ACA CCT CTG Cys Thr Pro Leu

CAG

Gin 325 ACC AGA ACT GTG Thr Arg Thr Val ATG CGG TTC CGA Met Arg Phe Arg TGC GAA Cys Glu 335 995 1043 1091 1139 1187 1235 1283 1336 1396 1456 1480 0*

S

S

S

*5S*

S

GAT GGA GAG Asp Gly Giu TGT AAC TAC Cys Asn Tyr 355

ATG

Met 340 TTT TCC AAG AAT GTC ATG ATG ATC CAG Phe Ser Lys Asia Val Met Met Ile Gin 34S TCC TGC AAA Ser Cys Lys 350 CGA CTG TAC Arg Leu Tyr AAC TGC CCG CAT Asia Cys Pro His

CCC

Pro 360 AAC GAG GCA Asia Glu Ala TCG TTC Ser Phe 365 AGC CTA Ser Leu 370 TTC AAT GAC ATC Phe Asia Asp Ile

CAC

His 375 AAG TTC AGG GAC Lys Phe Arg Asp TAAGTGCCTC CAGGGrI'CCT got.

*sees AGTGTGGGCT GGACAGAGGA GAAGCGCAAG CATCATGGAG ACGTGGGTGG GCGGAGGATG AATGGTGCCT TGCTCATI'CT TGAGTAGCAT TAGGGTATIT CAAAACTGCC

AAGGGGCTGA

TGTGGACGGA CAGCAGCGCA

GCCG

INFORM4ATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 379 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: misc feature OTHER INFORMATION: "Mouse Cyr~i amino acid sequence" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Ser Ser Ser Thr Phe Arg Thr Leu Ala Val A-ta Val Thr Leu Leu 1 5 10 His Leu, Thr Arg Leu Aia Leu Ser Thr Cys Pro Ala Ala Cys His Cys 2025 Pro Leu Glu. Ala Pro Lys Cys Ala Pro Gly Val. Gly Leu Val Arg Asp- 108 0009 0@ 0 0.* 0000 .00.

00000 0@ 8 0 0

OSOS

0 0000 0 Gly Cys Ser Lys Gly Ala Gly Arg Phe Gin Gly Cys .130 Cys Pro 145 Trp Vai Asp Leu Glu Leu Phe Gly 210 Gin Lys 225 Cys Gly J Arg Leu ~j Pro Val I 2 Lys Ser P 290 Lys Lys T 305 Cys Thr P Asp Gly G Cys Asn T' 3! Gly Thr Ser Pro Pro 115 Ile Asn Cys Gin Ser Cys 100 Asn Pro Pro Cys Pro Thr Glu Cys Leu Arg Ly Cy 7 Al Ty: LyI

CYE

Leu Cys Leu Ile L95 rhr -ys 'hr

T

al 'yr 75 ro yr ro 1u yr 55 Asr Gl 180 Ala Glu Ile Gly Lys 260 Ser Glu Arg Leu Met 340 Asn Glu 165 r Leu Ile Pro Val Ile 245 Glu Ser Pro Pro Gin 325 Phe Cys: 15c Asp Asp Gly Arg Gin 230 Ser Thr Leu Val Lys 310 Thr Ser Pro s Va

S

s As 0 Le As 3 Hi! Prc 13f Va Se2 Ala Lys Vai 215 Thr Thr Arg Lys Arg 295 Tyr Arg Lys His 40 .1 Cys

S

p His u Lys n Ser s Gin 120 Gin L Lys Ile Ser Gly 200 Leu Thr Arg Ile Lys 280 Phe I Cys C Thr Asn V 3 Pro A Ala Thr Gly Arg 105 Cys Glu Val Lys Glu 185 Ser Phe Ser Val .ys 265 'ly S Lhr '1 ral I 3 !al M 45 Leu Ser Asp 170 Val Ser Asn rrp rhr 250 3lu ys 'yr er lys 130 e t Thr Cys Ile Ser Gly 155 Ser Glu Leu Pro Ser 235 Asn Val Lys Ala Cys 315 Met Met Leu 140 Gin Leu Leu Lys Leu 220 Gin Asp Arg Cys Gly 300 Val krg Tie Lys Lys Ile 90 Ile Gin Leu Asn Glu As 60 Gly Leu GluCys Asl 75 Cys Arg Ala Gin Se 9' Tyr Gin Asn Gly Git 110 Asp 125 Pro Cys Asp Thr Arg 205 His Cys Asn Pro Ser 285 Cys Asp Phe 2 Gin Gly Asn Cys Asp Arg 190 Leu Ala Ser Pro Cys 270 Lys Ser 31

Y

krg er A1z Let Glu Gin 175 Asn Pro His Lys Glu 255 Gly Thr Ser Arg -ys 335 -ys p Cys n Phe Glu I Ser Val Gly Glu 160 Asp Asn Val Gly Ser 240 Cys Gin Lys Val Cys 320 Glu Lys 360 sn Giu Ala Ser Phe Arg Leu Tyr 365 Ser Leu Phe Asn Asp Ile His Lys Phe Arg Asp -109 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 1418 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY:

CDS

LOCATION: 124..1266 (ix) FEATURE: NAME/KEY: misc feature OTHER INFORMATION: "Human cyr6l cDNA coding sequence" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GGGCGGGCCC ACCGCGACAC CGCGCCGCCA CCCCGACCCC GCTGCGCACG

GCCTGTCCGC

TGCACACCAG CTTGTrGGCG TCTTCGTCGC CGCGCTCGCC CCGGGCTACT

CCTGCGCGCC

ACA ATG AGC TCC CGC Met Ser Ser Arg

ATC

Ile GCC AGG GCG CTC Ala Arg Ala Leu

GCC

Ala 10 TTA GTC GTC ACC CTT Leu Val Val Thr Leu CCC GCT GCC TGC CAC CTC CAC TTG ACC Leu His Leu Thr

AGG

Arg CTG GCG CTC TCC ACC TGC Leu Ala Leu Ser Thr Cys 25 Pro Ala Ala Cys His TGC CCC CTG Cys Pro Leu GAC GGC TGC Asp Gly Cys 50

GAG

Glu GCG CCC AAG TGC Ala Pro Lys Cys

GCG

Ala 40 CCG GGA GTC GGG Pro Giy Val Gly CTG GTC CGG Leu Val Arg AAC GAG GAC Asn Glu Asp C GGC TGC TGT AAG Gly Cys Cys Lys

GTC

Val 55 TGC GCC AAG CAG Cys Ala Lys Gin

CTC

Leu TGC AGC Cys Ser AAA ACG CAG CCC Lys Thr Gin Pro GAC CAC ACC AAG Asp His Thr Lys

GGG

Gly CTG GAA TGC AAC Leu Glu Cys Asn 168 216 264 312 360 408 456 504 552 600

TTC

Phe GGC GCC AGC TCC Gly Ala Ser Ser

ACC

Thr 85 GCT CTG AAG GGG Ala Leu Lys Gly

ATC

Ile 90 TGC AGA GCT CAG Cys Arg Ala Gln

TCA

Ser GAG GGC AGA CCC Glu Gly Arg Pro

TGT

Cys 100 GAA TAT AAC TCC Glu Tyr Asn Ser

AGA

Arg 105 ATC TAC CAA AAC Ile Tyr Gin Asn GGG GAA Gly Glu 110 AGT TTC CAG Ser Phe Gin GTG GGC TGC Val Gly Cys 130

CCC

Pro 115 AAC TGT CAA CAT Asn Cys Gin His

CAG

Gin 120 TGC ACA TGT ATT Cys Thr Cys Ile GAT- GGC GCC Asp Gly Ala 125 ATT CCT CTG TGT I-ie Pro Leu Cys CCC CAA Pro Gin 135 GAA CTA TCT CTC CCC AAC TTG Glu Leu Ser Leu Pro Asn Leu 140 GGC TGT CCC AAC CCT CGG CTG GTC AAA GTT ACC GGG CAG TGC TGC GAG 110- Giy Cys Pro Asn Pro Arg Leu Val Lys Val Thr Gly Gin Cys Cys Giu 145 150 155

GAG

Giu 160 TGG GTC TGT GAC Trp Vali Cys Asp

GAG-GAT

GJlu Asp 165 AGT ATC AAG Ser Ile Lys

GAC

Asp 170 CCC ATG GAG GAC Pro Met Giu Asp

GAG

Gin 175 GAC GGC CTC CTT Asp Gly Leu Leu

GGC

Giy 180 AAG GAG CTG GGA Lys Giu Leu Gly GAT GCC TCC GAG Asp Ala Ser Giu GTG GAG Val Glu 190 TCA CTG& Ser Leu 696 744 TTG ACG AGA Leu Thr Arg AAG CGG CTC Lys Arg Leu 210

AAC

Asn 195 AAT GAA TTG ATT Asn Giu .Leu Ile

GCA

Al a 200 GTT GGA AAA GGC Val Gly Lys Gly

AGA

Arg 205 CCT GTT T GGA Pro Val Phe Gly GAG CCT CGC ATC Giu Pro Arg Ile

CTA

Leu 220 TAC AAC CCT Tyr Asn Pro TTA CAA Leu Gin 225 GGC CAG AAA TGT Giy Gin Lys Cys

ATT

Ile 230 GT'r CAA ACA ACT Val Gin Thr Thr

TCA

Ser 235 TGG TCC GAG TGC Trp Ser Gin Cys

TCA

Ser 240 AAG ACC TGT GGA Lys Thr Cys Gly

ACT

Thr 245 GGT ATC TCC ACA Gly Ile Ser Thr

CGA

Axg 250 GTT ACC AAT GAC Val Thr Asn Asp

AAC

Asn 255 888 936 CCT GAG TGC CGC Pro Glu Cys Arg

CTT

Leu 260 GTG AAA GAA ACC Val Lys Giu Thr

CGG

Arg 265 ATT TGT GAG GTG Ile Cys Giu Val CGG tCT Arg Pro 270 TGT GGA GAG Cys Gly Gin AAG ACC AAG Lys Thr Lys 290

CCA

Pro 275 GTG TAC AGC AGC Val Tyr Ser Ser

CTG

Leu 280 AAA AAG GGC AAG Lys Lys Gly Lys AAA TGC AGC Lys Cys Ser 285 GCT GGA TGT Ala Giy Cys AAA TCC CCC GAA Lys Ser Pro Giu

CA

Pro 295 GTC AGG TTT ACT Val Arg Phe Thr

TAC

Tyr 300 TTG AGT Leu Ser 305 GTG AAG AAA TAC Val Lys Lys Tyr

CGG

Arg 310 CCC AAG TAC TGC Pro Lys Tyr Cys

GGT

Gly 315 TCC TGC GTG GAC Ser Cys Val Asp

GGC

Gly 320 CGA TGC TGC ACG Arg Cys Cys Thr

CCC

Pro 325 CAG CTG ACC AGG Gin Leu Thr Arg

ACT

Thr 330 GTG AAG ATG CGG Val Lys Met Arg

TTC

Phe 335 984 1032 1080 1128 1176 1224 1266 1326 1386 CGC TGC G.AA GAT Arg Cys Giu Asp

GGG

Giy 340 GAG ACA TTT TCC Giu Thr Phe Ser AAC GTC ATG ATG Asn Val Met Met ATC GAG Ile Gin 350 TCC TGC AAA Ser Cys Lys CCC TTC-TAC Pro Phe Tyr 370

TGC

Cys 355 AAC TAC AAC TGC Asn Tyr Asn Cys

CCG

Pro 360 CAT GCC AAT His Ala Asn GAA GGA GCGTT Giu Ala Ala Phe 365 AGG CTG TTC AAT Arg Leu Phe Asn

GAC

Asp 375 ATT CAC AAA 'TT Ile His Lys Phe AGG GAC Arg Asp 380 TAAATGCTAC CTGGGTTTCC AGGGCACACC TAGACAAACA AGGGAGAAGA GTGTCAGAAT CAGAATCATG GAGAAAATGG GCGGGGGTGG TGTGGGTGAT GGGACTCATT

GTAGAAAGGA

1.1 1 AGCCTTCTCA TrCTTGAGGA GCATrAAGGT AT.

INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 381 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: misc feature OTHER INFORMATrON:* "HPuman CyrG1 amino acid sequence" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 1418 Met 1 Ser Ser Arg Ile Ala Arg Ala Leu A 4* His Leu Thr Arg 20 Leu Ala Leu Pro Leu Giu Ala Pro Lys Cys 35 Gly Cys Gly Ser Gly Gly Phe Gly Cys 145 Trp Gly Thr Arg Gin 225 50 Lys Ala Arg Gin Cys 130 Pro Val Leu Arg Le u 210 Gly Thr Ser Pro Pro 115 Ile Asn Cys Leu Asn 195 Pro Gin Cys Cys Lys Gin Pro Cys 70 Ser Thr Ala Cys Giu Tyr 100 Asn Cys Gln Pro Leu Cys Pro Arg Leu 150 Asp Glu Asp 165 Gly Lys Glu 180 Asn Giu Leu Val Phe Gly Lys Cys Ile Val 55 Asp Leu Asn His Pro 135 Val Ser Leu Ile Met 215 Val Ser Ala 40 Cys His Lys Ser Gln 120 Gin Lys Ile Gly Ala 200 Glu Gln Thr 25 Pro Ala Thr Gly .Arg 105 Cys Giu Val Lys Phe 185 Vai Pro rhr

C

G

L

L

T]

Ti Gl Az Th ~la Leu Val 10 'ys Pro Ala ly Val Gly ys Gin Leu ys Gly Leu 75 le Cys Arg 90 le Tyr Gin hIr Cys Ile eu Ser Leu 140 -ir Giy Gin 155 ;p Pro Met 70 p, Ala Ser .y Lys Gly *g Ile Leu 220 Lr Ser Trp 235 Val Al a Leu Asn Giu Al a Asn Asp 125 Pro- Cys Giu Giu Arg 205 Tyr Ser.

*Thr *Cys Val Giu Cys Gln Gly 110 Gly Asn Cys Asp Val 190 Ser Asn Gi n Leu is His Arg Asp Asn Ser Giu Ala Leu Giu Gln 175 Giu Leu Pro Cys Leu Cys Asp Cys Phe Glu Ser Val Gly Glu 160 Asp Leu Lys Leu- Ser 240 230 Lys Thr Cys Gly Thr Gly Ile Ser Thr Arg Val Thr Asn Asp Asn Pro 112 245 Val.

Giu Cys Arg Leu 260 Giy Gin Pro Val 275 Thr Lys Lys Ser Lys Giu Thr Arg 265 Lys 250 Ile Lys 255 Tyr Ser Ser Leu 280 Val.

Cys Glu Val. Arg Pro Cys 270 Giy Lys Lys Cys Ser Lys 285 Thr Tyr Ala Giy Cys Leu Pro Giu 290 Ser Vai Pro 295 Pro Arg Phe 300 Ser Lys Lys Tyr 305 Arg Arg 310 Gin Lys Tyr Cys Gly 315 Val Cys Val. Asp Gl 320 Cys Cys Thr Leu Thr Arg Thr 330 Asn Lys Met Arg Phe Arg 335 Cys Giu Asp Giy 340 Thr Phe Ser Lys 345 His Val met Met Ile Gin Ser 350 Aia Phe Pro Cys Lys Cys Asn Tyr Asn Cys 355 Phe Tyr Arg Leu Phe Asn Asp 370 375 INFORMATION FOR SEQ ID N~ Pro 360 Ile Aia Asn Glu Al a 365 Asp His Lys Phe Arg 380 0O: SEQUENCE CHAR.ACTERISTICS: LENGTH: 2267 base pairs TYPE: nucleic acid STR.ANDEDNESS: single TOPOLOGY: linear (iMOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: misc feature OTHER INFORMATION: "Fispi2 cDNA coding sequence,, (xi) SEQUENCE DESCRIPTION: SEQ ID GAATTCCGCC GACAACCCCA GACGCCACCG CCTGGAGCGT

CTGTCCGAAT

CCTACCGCGT

TCCTCGCCCT

CAGCCGAAGC

GCTGCCGCGT

CACACAAGGG

CTGCCAAAGA

TCCAAAGCAG

TATGCAGCAT

CCAGGCTCCA

CCCGATCATG

CTGCACCCGG

AGCGCCGCAC

CTGCGCCAAG

CCTCTTCTGC

TGGTGCACCC

CTGCAAATAC

GGACGTGCGC

GCCGCGCCTC

CTCGCCTCCG

CCTGCTACGG

TGCCCCGCCG

CAGCTGGGAG

GATTTCGGCT

TGTGTCTTCG

CAATGCACTT

CTGCCCAGCC

TCGTCGCCTC

TCGCAGGTCC

GCCAGGACTG

GCGTGAGCCT

AACTGTGTAC

CCCCCGCCAA

GTGGGTCGGT

GCCTGGATGG

CTGACTGCCC

CCAGACACCA

TGCACCCTGC

CATCAGCCTC

CAGCGCGCAA

GGTGCTGGAC

GGAGCGTGAC

CCGCAAGATT

GTACCGCAGC

GGCCGTGGGC

CTTCCCGAGA

ACCTCCGCCC

TGTGCATCCT

GCCTTGGTGC

TGTCAGTGCG

GGCTGCGGCT

CCCTGCGACC

GGAGTGTGCA

GGTGAGTCCT

TGCGTGCCCC

AGGGTCAAGC

120 180 240 300 360 420 480 540 600 TGCCTGGGAA ATGCTGCAAG GAGTGGGTGT GTGACGAGCC CAAGGACCGC ACAGCAGTTG 113 GCCCTGCCCT AGCTGCCTAC CGACTGGAAG ACAC.ATTT.GG -CCCAGACCCA

ACTATGATGC

CC..

GAG CCAACT

GCATCTCCA,

TCTGCATGG'

GCATCCGGA(

TGAAGACAT

ACAGAACCAC

ATATGATGrn

TTGAGTCCCI

ACACGAACTC

AATTACAGTP

ACCAAAGTGP

TTCGAGACAG

GGTGAGTCTC

TATTTTAAAA

ATGCTTGC.AG

TGAGTCCAGC

CACTTGTCAG

CCTGTAAC.AA

TATATTTGTA

GGGATTTTAA

GTGATTCAAA

AACAAACAAA

AATGTGGTCA

GTAAGGTCCG

TGGGGGGCAG

TGAAATA

G CCTGGTCCAi C CCGAGITACi r CAGGCCCTG( :ACCTAAAATc k. CAGGGCTAA(

"CACTCTGCC;

7CATCAAGACC 7GTACTACAGC 'ATTAGACTA7r LGCAC.ATTAA7

GAACGTTATG

TTTACACTTG

CTGG.AACAGT

GCAGCAGTGT

.ACAGACCTGC

TGTTCTTTAA

GAGTCAGAGC

GCCAGArTT CAG'rrATCTA

ACTGATAGCC

ACAAAGGAGA.

TGCTGTGCAG

AATCCCTGTT

ATTCCTACCA

TTTATTTGTT

GTATATATAT

3ACCACAGAGJ AATGACAATz

:G.AAGCTGACC

GCC.AAGCCTC

TTCTGCGGGC

kGTGGAGTTCA

TGTGCCTGCC

AAGATGTACG

AACTTGAACT

TT.AAATCTGT

TCATGGCCAT

ACAGTTGTTC

GGAGATGCCA

GCCTACTTTT

TCTAGCGAGA

GAACAGCAGT

CTTGTCTGTt

TATTGATAT

AGTTAATA

TCAAACTCCA

TACTGCAGTG

GTGATAAAGC

GGTGAACAAA

GGAAGTGCTT

GAGAGTGTGA

7GGAGCGCCT( k CCTTCTGCA( *TGGAGGAAA1

TCAAGTTTGJ

TGTGCACAGz

*AATGCCCCGX

ATTACAACTCO

GAGACATGGC

GAGTTGCATC

GI1TI'TAACT

ACAAGTAGTC

ATTAGCGCAC

GGAGAAAGAA

TGGAGTGTAA

GCTGAGCATG

TTCAGCCTCT

AGACTGGACA

GTAAATATTG

AAGTCATTTG

AACACCATAG

GGAATI'GTGA

TATGTATTGG

TGGCCTTTAT

GCTGCTTCTT

CCAAAAGTTA

;ACTGGAGAA(

kCATTAAGAAC kGCTTTCTGG(

LCGGCCGCTGC

LTGGCGAGATC

FTCCTGGGG.AC

GTAAAGCCAG

TCATTTTCTI

ACCGTGGGAG

TGTCAACCTC

AGTGCCAGAA

AGACAGGTAC

CCGGGGAGGG

TGTCCTCCAC

GACCATTCTG

GCTTGTGGCA

TGGATATATA

TTTTTGTIT

GTAGGACACG

CCTGAGTGAC

AAGTCAGATT

TAAGAAATGG

TGATTATGAC

CATGTTTGCA

3CAGAGCCGCC

GGCAAAAAGT

TGCACCAGTG

TGCACACCqC

ATGAAAAAGA

AATGACATCT

FGAAGTAAGGG

CTGTAAAAAc

GAACTATCCC

AGACACTGGT

CGCACACTGA

TAGCTGAGGT

AAATTATAGC

TAGATGAGGC

ATTCCAGTGA

.AGTAAGTTTG

TATATATATA

AAGTGCTTTT

AAGCTTATCT

TCTCTGTCAG

TCTAGTAGGA,

CTGGCTCAGG

TGGTTTGGGG

CCTTTCTAGT

TTCTAAGACC TGTGGAATGG 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2267 ATTTTTTATA TGAAAAAA

GGAATTC

INFORMATION FO. SEQ ID NO:6: SEQUENCE

CHARACTERISTICS:

LENGTH: 348 amino acids TYPE: amino acid STR-aNDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein 114- (ix) FEATURE: NAME/KEY::misc -feature OTHER INFORMATION: "Fisp12 amino acid (xi) SEQUENCE D. WRI2IODL-S.QIfbNO. sequence" Met Leu Ala Ser Val Ala Gly Pro Ile Ser Leu Ala Leu Val Leu Leu 1 5 10 Ala Gin Vai Glu Cys Lys Glu Ala Pro 145 Lys Ala Met I Ser I Thr 1 225 Cys C Arg T Thr S Gly A 2 Let Cye Lei Le.

Asp Asp Ser Val 130 Asp Glu Leu 4e t io ys 'he lu 'hr er rg 90 .1 Cyr 3 Al i AsE 1 Cys Phe I Gly Phe Gly Cys Trp Ala Arg 195 Thr Cys Ala Pro Val .275 Cys s Thr Arg i Ala Glu Gly Cys Thr Glu Gly Ser *Ala Pro 100 Gin Ser Cys Val Pro Phe Val Cys 165 Ala Tyr 180 Ala Asn Cys Gly i Arg Leu Asp Leu 245 Lys Ile 2 260.

Lys Thr 'J Cys Thr E Al Gil Arc 70 Prc Cys Ser Pro Pro 150 Asp Arg .ys Met lu 230 3lu i a .yr 'ro a 25 Ala Pro His Cys Pro r Cys 55 Asp Ala Vai Cys Leu 135 Arg Glu Leu Leu Gly 215 Lys Glu Lys Arg His 295 40 Cys Pro Asn Phe Lys 120 Cys Arg Pro Glu Val 200 Ile Gin Asn Pro Ala 280 Arg Arg Cys Arg Gly 105 Tyr Ser Val Lys Asp 185 Gln Ser Ser Ile Val 265 Lys Thr Val Asp Lys 90 Gly Gin Met Lys Asp 170 Thr Thr Thr Arg Lyes 250 Lys Phe trhr Cys Pro 75 Ile Ser Cys Asp Leu 155 Arg Phe Thr Arg Leu 235 Lys Phe Cys Thr Ala Ala His Gly Val Thr Val 140 Pro Thr Gly Glu Val 220 Cys 1 Gly Glu Gly Leu 1 300 Gl Lys Lys Val Tyr Cys 125 Arg Gly Ala Pro rrp 205 rhr .4e t ys eu lai ro Val Gin Gly Cys Arg 110 Leu Leu Lys Val Asp 190 Ser Asn Val Lys 4 Ser 270 Cys Val Ser Leu Leu Thr Ser Asp Pro Cys Gly 175 Pro Ala Asp Arg :ys 255 Gly rhr 3lu Leu Gly Phe Ala Gly Gly Ser Cys 160 Pro Thr Cys Asn Pro 240 Ile Cys Asp Phe Pro Ala Thr Gly Gin Asp Cys Ser Ala Gin Cys -115- Lys Cys Pro Asp.Gly Glu Ile Met Lys Lys Asn Met Met Pile Ile Lys 305 310 315 320 Thr Cys Ala Cys His Tyr Asn Cys Pro Gly ASP Asn Asp Ile Pile Glu 325 330 335 Ser Leu Tyr Tyr Arg Lys Met Tyr Gly Asp Met Ala 340 345 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: CA) LENGTH: 2075 base pairs TYPE: nucleic acid STRANDEDNESS: single' TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: misc feature OTHER INFORMATION: "1CTGF cDNA coding sequence"' (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CCCGGCCGAC AGCCCCGAGA CGACAGCCCG GCGCGTCCCG

CCAGCGCTCC

GTGCCAACCA

GCCCTCTGCA

GAGCCGGCGC

CGCGTCTGCG

AAGGGCCTCT

AAAGATGGTG

AGCAGCTGCA

AGCATGGACG

GGGAAATGCT

GCCCTCGCGG

AACTGCCTGG

TCCACCCGGG

ATGGTCAGGC

CGTACTCCCA

ACATACCGAG

ACCACCACCC

ATGTTCATCA

AGGCCCCGCG

TGACCGCCGC

GCCGGCCGGC

CGCGCTGCCC

CCAAGCAGCT

TCTGTGACI'

CTCCCTGCAT

AGTACCAGTG

TTCGTCTGCC

GCGAGGAGTG

CTTACCGACT

TCCAGACCAC

TTrACCAATGA

CITGCGAAGC

AAATCTCCAA

CTAAATTCTG

TGCCGGTGGA

AGACCTGTGC

CTCCCCGCTC

CAGTATGGGC

CGTCGGCCAG

GGCGGGCGTG

GGGCGAGCTG

CGGCTCCCCG

CTTCGGTGGT

CACGTGCCTG

CAGCCCTGAC

GGTGTGTGAC

GGAAGACACG

AGAGTGGAGC

CAACGCCTCC

TGACCTGGAA

GCCTATCAAG

TGGAGTATGT

GTTCAAGTGC

CTGCCATTAC

GCCGCCACCG

CCCGTCCGCG

AACTGCAGCG

AGCCTCGTGC

TGCACCGAGC

GCCAACCGCA

ACGGTGTACC

GACGGGGCGG

TGCCCCTTCC

GAGCCCAAGG

TTTGGCCCAG

GCCTGrI'CC.A

TGCAGGCTAG

GAGAACATTA

TTTGAGCTTT

ACCGACGGCC

CCTGACGGCG

AACTGTCCCG

GTCCCCACCT

CGCCCTCCGC

TCGCCTTCGT

GGCCGTGCCG

TGGACGGCTG

GCGACCCCTG

AGATCGGCGT

GCAGCGGAGA

TGGGCTGCAT

CGAGGAGGGT

ACCAAACCGT

AC CCAACTAT

AGACCTGTGG

AGAAGCAGAG

AGAAGGGCAA

CTGGCTGCAC

G.ATGCTGCAC

AGGTCATGAA

GAGACAATGA

CCGACCACCG

TCCGCCCGCA

GGTCCTCCTC

GTGCCCGGAC

CGGCTGCTGC

CGACCCGCAC

GTGCACCGCC

GTCCTTCCAG

GCCCCTGTGC

CAAGCTGCCC

GGTTGGGCCT

GATTAGAGCC

GATGGGCATC

CCGCCTGTGC

AAAGTGCATC

CAGCATGAAG

CCCCCACAGA

GAAGAACATG

CATCTTTGAA

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 116 TCGCTGTACT ACAGGAAGAT GT.ACGGAGAC ATGGCATGAA GCCAGAGAGT GAGAGACATT

S

AACTCATTAG ACTGGAACTI

GTAGCACAAG.TTATTTAAAT.

AAACATTGTG CCATGTCAAA TAAGACTTGA CAGTGGAACT TTTAGGAGCA GTGGGAGGGT TAATATGCCT GCTAT'TTGAA GCCTGTAGCC CCAGTGACAG GTTGTTCCTT AAGTCAGAAC TCAGGAATCG GAATCCTGTC ACAAGCCAGA T TTT TTAAAA TATATATATA TATGTACAGT TGTTTTTAAT GCTTTGATAT GTAAAGCTTG TCTGATCGTT ATAGAATGAC AGTCCGTCAA GGCTGATTTC TAGGTAGGAA CAAATAGTCT ATCTTCCCCA GACACTGGTT ACATTAGTAC ACAGC ACCGGCCCGG TTAGT GTGTAATTGA GAAGG CTAGGATGTG CATTC

AGC-AGACTCA.GCTCT

GATTAGACTG GACAG TTTATATTGT AAATA' TATCTAAGTT AATTT TTCAATGTTA GCCTC CAAAGCATGA AATGG AACAGATTGT TTGCAj ATGTGGTAGC TCACG

ACCAG

ATCAT

AAAAT

TCCAG

GACAT

CTTGT

~TGTG

k.AAGT

%ATTT

WTACT

AATGTATATT

CAGATCGACT

TTTAGCGTGC

CCATCAAGAG

TCTGATTCGA

GGCAAGTGAA

TGTGTGTGTG

TGTTTGTGCC

CTGAACACCA

TATATGGAAA

TGAAGAATGT

AAGGTGTGGC

CTTATACGAG

TCACTGACCT

ACTGAGTCAA

ATGACACTGT

ITTGCCTGTA

TGTGTGTATA

TITTAFF'IW

TAGGTAGAAT

TTCTGCTCAG

GAACTGATTC ACATCTCATT TTTCCGTAAA AATGATTTCA £I~.CTAACTGGGGGGA AGATICCC aCCCAATTCA 1200.

1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2075 ~GGG GAGGCATCAG TGTCTTGGCA INFORMATION FOR SEQ ID NO:8:

S.

SEQUENCE CHARACTERISTICS: LENGTH: 349 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY:'linear ii) MOLECULE TYPE: protein ix) FEATURE: NAME/KEY: misc feature OTHER INFORMATION: '"CTGF amino acid sequence" xci) SEQUENCE DESCRIPTION: SEQ ID NO:8: MIet Thr Ala Ala Ser Met Gly Pro Val Arg Val Ala Phe Val.

1 5 10 Val Leu Leu Ala Leu Cys Ser Arg Pro Ala Val Gly Gin Asn Cys Ser Gly Pro 25 Cys Arg Cys Pro Asp Glu Pro Ala Pro Arg Cys Pro Ala Gly Val Ser 40 Leu Val Leu Asp Gly Cys Gly Cys Cys Arg Val Cys Ala Lys Gin Leu 55 Gly Glu Leu Cys TI-r Glu Arg- Asp Pro Cys Asp Pro His Lys Gly Leu 70 75 an 117 Phe Cys Asp Phe Gly Ser Pro Ala Asn Arg Lys Ile Gly Val Cys Thr 90 Al a Gly Gly Lys Glu Al a Asp Ser 115 Val Gly 100 Phe Gly Al a Gln Cys Prc Ser Met 130

S.

C

*5

S

Ser 145 Cys Pro Thr Cys Asn 225 Pro -Ile Cys Asp Phe 3.05 Lys Glu Ala Me t Ser 210 Al a Cys Arg Thr Gly 290 Lys rhr Glc Leu Ile 195 Lys Ser Glu Thr Ser 275 Arg Cys Cys Trp Ala 180 Arg Thr Cys Al a Pro 260 Met Cys Pro Ala Tyr 340 Val 165 Al a Al a Cys Arg Asp 245 Lys Lys Cys Asp 325 150 Cys Tyr Asn Gly Leu 230 Leu Ile Thr Thr Gly 310 His Pro Asp Cys Pro Phe Cy Se~ Prc 135 Pro Asp Arg Cys Met 215 Glu Glu Ser Tyr Pro 295 Glu Tyr sIlE *Cys 120 Leu Arg Glu *Leu Leu 200 Gly Lys Giu Lys Arg 280 His Val Asn Met Phe 105 Lys Cys Arg Pro Glu 185 Val Ile Gln Asn Pro 265 Ala Arg Met Cys TyrC 345 Gl~ Tyi Sex Val Lys 170 Asp Gln Ser Ser Ile 250 I le Lays rhr Lys P ro 330 31Y G.1y Thr Val Tyr Arg Ser 110 Gln Cys Thr Cys Leu Met *Lys 155 Asp Thr Thr Thr Arg 235 Lys Lys Phe Thr Lys 315 Gly.

Asp Asp 140 Leu Gin Phe Thr Arg 220 Leu Lys Phe Cys Thr 300 Asn Asp Met 125 Val Pro Thr Gly Glu 205 Val Cys Gly Glu Gly' 285 Leu Met Asn Ala Arg Gly Val Pro 190 Trp Thr Met Lys Leu 270 Val Pro 4'sp Leu Lys Val 175 Asp Ser Asn Val Lys 255 Ser Cys Val Phe Ile 335 Asp Pro Cys 160 Gly Pro Ala Asp Arg 240 Cys Gly Thr Giu Ile 320 Phe Glu Ser Leu Tyr Arg Lys INFORMATION FOR SEQ ID NO:9: Wi SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDN'A 118 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: GGGGATCTGT GACGAGCCCA AGGAC INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID GGGAATTCGA CCAGGCAGTT GGCTCG 26 INFORMATION FOR SEQ ID NO:ll: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll: GGGGATCCTG TGATGAAGAC AGCATT 26 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: GGGAATTCAA CGATGCATTT -CTGGCC 26 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: (A)-LENGTH: 21 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant 119 (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: Asp Gly Cys Gly Cys Cys Lys Val Cys Ala Lys Gin Leu Asn Glu Asp 1 5 10 Cys Ser Lys Thr Gin INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 21 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: Pro Asn Cys Lys His Gin Cys Thr Cys Ile Asp Gly Ala Val Gly Cys 1 5 10 Ile Pro Leu Cys Pro INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 24 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Cys Ile Val Gin Thr Thr Ser Trp Ser Gin Cys Ser Lys Ser Cys Gly 1 5 10 Thr Gly Ile Ser Thr Arg Val Thr INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 26 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant 120- (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2.S: Ile Ser Thr Arg Val Thr Asn Asp Asn Pro Glu Cys Arg Leu Val Lys 1. 5 10 Glu Thr Arg Ile Cys Glu Val. Arg Pro Cys INFORM4ATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 21 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide 0 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: Lys Tyr Cys Gly Ser Cys Val Asp GJly Arg Cys Cys Thr Pro Leu Gin 1o~ 5 10 is Thr Arg Thr Val Lys .0.0* 121

Claims (2)

122- THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:- 1. An antibody that specifically binds to a polypeptide having the amino acid sequence set forth in SEQ ID NO:4, and analogs and/or derivatives of said polypeptide. 2. The antibody of claim 1 wherein said antibody binds within amino acid residues 163 to 229 of SEQ ID NO: 4. 3. The antibody of claim 1 or claim 2 wherein said antibody binds within amino acid residues 170 to 185 of SEQ ID NO:4. 4. The antibody of any one of claims 1 to 3 wherein said antibody is an antibody fragment. 5. A recombinant antibody that specifically binds to a polypeptide having the amino acid sequence set forth in SEQ ID NO: 4, and analogs and/or derivatives of said polypeptide. 6. The antibody of claim 5 wherein said antibody binds within amino acid residues 163 to 229 of SEQ ID NO: 4. 7. The antibody of claim 6 wherein said antibody binds within amino acid residues 170 to 185 of SEQ ID NO: 4. S. 8. The antibody of any one of claims 5 to 7 wherein said antibody is a chimeric antibody, a humanized antibody, or a CDR-grafted antibody. S* 9. The antibody of any one of claims 5 to 8 wherein said antibody is an antibody fragment. The antibody of any one of claims 1 to 9 wherein said antibody is attached to a label. 11. The antibody of claim 10 wherein said label is selected from the group consisting Sof signal-generating enzymes, antigens, antibodies, lectins, carbohydrates, biotin, avidin, S* 25 radioisotopes, toxins and heavy metals.

123- 12. The antibody of any one of claims 1 to 9 wherein said antibody is attached to a toxin. 13. The antibody of claim 12 wherein said toxin is ricin. 14. An antibody that specifically binds to a polypeptide having the amino acid sequence set forth in SEQ ID NO: 4, substantially as herein described with reference to any one of the examples excluding comparative examples. A recombinant antibody that specifically binds to a polypeptide having the amino acid sequence set forth in SEQ ID NO: 4, substantially as herein described with reference to any one of the examples excluding comparative examples. DATED this 24th day December, 2003 BALDWIN SHELSTON WATERS Attorneys for: MUNIN CORPORATION